From Byproduct to Resource: Fermented Apple Pomace as Beer Flavoring

One of the main struggles of the large-scale apple processing industry is pomace disposal. One solution for this problem is to convert this waste into a resource. Apple pomace could be used as a substrate for lactic acid bacteria and could induce the formation of a more complex aroma profile, making this fermented product an innovative aromatizer for alcoholic beverages, such as beer. In this study, for the first time, the effect of lacto-fermented apple pomace addition in beer was evaluated. Three bacterial strains (Lactobacillus rhamnosus 1473 and 1019, and L. casei 2246) were tested for apple pomace fermentation, and L. rhamnosus 1473 was the strain that best modified the aromatic profile. The addition of fermented apple pomace to beer increased the complexity of the aroma profile, demonstrating the potential of this byproduct as an aromatizer in the alcoholic beverage industry.


Introduction
With almost 90 million tons produced all over the world in 2016, apple (Malus domestica Borkh.) is one of the most cultivated fruit crops [1], and it can be consumed fresh or as juice, cider, wine, and vermouth, purees, jams, and dried products [2].
The apple transformation industry generates large amounts of waste known as 'apple pomace' (3600 ktons in 2010), which is mainly composed of skin and flesh (95%), seeds (2% to 4%), and stems (1%) [3]. For the large-scale apple processing industry, pomace disposal is a big problem, impacting massively on the environment; for this reason, in recent years, many efforts have been made to convert this waste into a resource, exploiting its richness in nutrients. Indeed, apple pomace composition varies depending on the variety, origin, and processing technology prior to its generation, but in general, it contains 70% water, 16% carbohydrates, 7% cellulose, 5% protein [4], and many valuable polyphenols with antioxidant properties [5]. Often, only 20% is retrieved as animal feed and the remaining 80% goes to landfill, is incinerated, or is sent to composting sites which results in the release of greenhouse gases [6]. On the contrary, apple pomace waste is an excellent resource for the production of various chemical compounds, such as pectin, limonene, and d-galacturonic acid [7]; moreover, thanks to its abundance of fermentable soluble sugars, including glucose, apple pomace is a perfect substrate for fermentative processes.
In order to valorize and to find a different use for byproducts obtained by the apple processing industry, the effect of lacto-fermented apple pomace addition to beer has been evaluated for the first time in this study. To reach this main objective, the following aspects were investigated: (i) evaluating the use of three lactic acid bacteria (LAB) strains to ferment apple pomace; (ii) evaluating, per each LAB, the effect on the aromatic profile of apple pomace; (iii) evaluating the effect of a thermal treatment on the volatile profile of unstarted and fermented apple pomace; (iv) evaluating the effect on beer aromatic profile of lacto-fermented apple pomace addition. protocol reported by Ricci et al. (2018) [8]. Samples of 2 g of apple pomace or 2 mL of beer were used for the analyses, adding 10 µL of an aqueous toluene standard solution (100 µg/mL), useful for the semi-quantification of all detected signals. Head space micro-extraction was performed using a SPME fiber coated with three polymers of different polarity, divinylbenzene-carboxen-polydimethylsiloxane (Supelco, Bellefonte, PA, USA). The analyses were performed on a Thermo Scientific Trace 1300 gas chromatograph coupled to a Thermo Scientific ISQ single quadrupole mass spectrometer equipped with an electronic impact (EI) source (Thermo Fisher Scientific, Waltham, MA, USA), and the separation of analytes was achieved on a SUPELCOWAX 10 capillary column (Supelco, Bellefonte, PA, USA; 30 m × 0.25 mm × 0.25 µm). The characteristic volatiles were identified both by comparing mass spectra with the library (NIST 14) and by calculating their linear retention indices (LRIs).

Statistical Analysis
One-way ANOVA and two-way ANOVA were used to evaluate differences among treatments on apple pomace and on beers; mean separation was performed with Tukey's test (p ≤ 0.05) (SYSTAT 13.1, Systat Software, Inc., Pint Richmond, CA, USA).
In order to verify analogies and differences in beer profiles on the basis of their ingredients and maturation time, the data obtained from volatile profiles determination were statistically elaborated by SPSS Statistics 23.0 software (SPSS Inc., Chicago, IL, USA). In particular, principal component analyses (PCA) were carried out using as variables the concentrations of each volatile detected in the aromatic profiles of the different beer samples. PCA were performed using a covariance matrix and two factors were extracted.

Apple Pomace Fermentation
In order to evaluate the microbial contamination of apple pomace, total microbial count was performed. Since it was 4.14 ± 0.04 Log CFU/g, apple pomace was subjected to sterilization and used assubstrate for the further fermentation process. To this aim, three different strains belonging to L. rhamnosus (1473 and 1019) and L. casei (2246) species were employed. Results highlighted that all the tested strains were able to grow in apple pomace, increasing their concentrations of 2.52 Log CFU/g for L. rhamnosus 1473, 2.66 Log CFU/g for L. rhamnosus 1019, and 2.44 Log CFU/g for L. casei 2246 in 72 h. The similarities in the growth ability may be due strictly to the genotype-phenotype relationship. Fungi and yeast have been previously used for apple pomace fermentation, producing different compounds such as enzymes [6], citric acid [9], and animal feed [10]. To the best of our knowledge, lactic acid bacteria, mainly employed for lactic acid production [11], have never been used for apple pomace fermentation.

Effect of Different LAB Strains on Volatile Profile of Fermented Apple Pomace
A total of 45 different compounds were detected in the headspace of all samples and their identification was reported in Table 1. The main classes observed were esters, ketones, terpenes, terpene derivatives, and norisoprenoids and alcohols. The highest significant concentration of total volatile compounds was observed in apple pomace fermented with L. rhamnosus 1473 (21.03 ± 1.55 µg/mL) (Figure 1). This strain showed the statistically highest amount of hexanol, previously reported after L. plantarum [12] and L. rhamnosus [8] fermentation. Among the other detected alcohols, 2-hexenol meaningfully improved its concentration after the employment of 1473 strain. These data, in accordance with an earlier study [8], confirmed that L. rhamnosus 1473 is able to improve the quantity of these alcohols in different fruit matrices.
Esters were one of the most representative classes observed in apple pomace fermented with L. rhamnosus 1473, increasing their concentration by about seven times compared to the control. Among them, butyl 2-methylbutanoate, hexyl acetate, hexyl n-valerate, and hexyl caproate were the most concentrated compounds. Butyl 2-methylbutanoate a fatty acid ester with a typical apple aroma [26] increased only after L. rhamnosus 1473 fermentation and, at the same time, hexyl acetate, found in apple pomace, was preserved only during the fermentation with this strain.. Indeed, in the sample started with L. rhamnosus 1473, the resulting amount of hexyl acetate was statistically comparable with the control sample; by contrast, the samples started with L. casei 2246 contained a significantly lower concentration of this compound. Hexyl n-valerate and hexyl caproate, typical apple compounds, showed an increase when L. rhamnosus 1473 was used. Among aldehydes, benzaldehyde was the most concentrated, especially after L. rhamnosus 1473 fermentation, showing a statistically different amount from the other samples (Table S1). Generally, the enzymatic conversion of phenylalanine led to the formation of phenyl pyruvate, and then benzaldehyde was formed by a non-enzymatic step [27].  Esters were one of the most representative classes observed in apple pomace fermented with L. rhamnosus 1473, increasing their concentration by about seven times compared to the control. Among them, butyl 2-methylbutanoate, hexyl acetate, hexyl n-valerate, and hexyl caproate were the most concentrated compounds. Butyl 2-methylbutanoate a fatty acid ester with a typical apple aroma [26]increased only after L. rhamnosus 1473 fermentation and, at the same time, hexyl acetate, found in apple pomace, was preserved only during the fermentation with this strain.. Indeed, in the sample started with L. rhamnosus 1473, the resulting amount of hexyl acetate was statistically comparable with the control sample; by contrast, the samples started with L. casei 2246 contained a significantly lower concentration of this compound. Hexyl n-valerate and hexyl caproate, typical apple compounds, showed an increase when L. rhamnosus 1473 was used. Among aldehydes, benzaldehyde was the most concentrated, especially after L. rhamnosus 1473 fermentation, showing a statistically different amount from the other samples (Table S1). Generally, the enzymatic conversion of phenylalanine led to the formation of phenyl pyruvate, and then benzaldehyde was formed by a nonenzymatic step [27].
Comparing all the started samples and unfermented apple pomace, L. rhamnosus 1473 was the only one showing an increase in total ketone amount, especially sulcatone, which is a flavoring compound found in citrus fruit [29]. Its implementation was observed by Wang, Zhang, Li, & Xu (2013) [30] in grape extract, treated with β-glucosidase, but its increase has never been described after lactic acid fermentation.
Overall, based on these data, L. rhamnosus 1473 was the strain that best maintained and/or increased the characteristic aromatic compounds of apple. For this reason, lacto-fermented apple pomace with L. rhamnosus 1473 was chosen and used as a beer flavoring agent. Esters were one of the most representative classes observed in apple pomace fermented with L. mnosus 1473, increasing their concentration by about seven times compared to the control. Among m, butyl 2-methylbutanoate, hexyl acetate, hexyl n-valerate, and hexyl caproate were the most centrated compounds. Butyl 2-methylbutanoate a fatty acid ester with a typical apple aroma ]increased only after L. rhamnosus 1473 fermentation and, at the same time, hexyl acetate, found in ple pomace, was preserved only during the fermentation with this strain.. Indeed, in the sample rted with L. rhamnosus 1473, the resulting amount of hexyl acetate was statistically comparable th the control sample; by contrast, the samples started with L. casei 2246 contained a significantly er concentration of this compound. Hexyl n-valerate and hexyl caproate, typical apple pounds, showed an increase when L. rhamnosus 1473 was used. Among aldehydes, benzaldehyde s the most concentrated, especially after L. rhamnosus 1473 fermentation, showing a statistically ferent amount from the other samples (Table S1). Generally, the enzymatic conversion of enylalanine led to the formation of phenyl pyruvate, and then benzaldehyde was formed by a nonzymatic step [27].
Comparing all the started samples and unfermented apple pomace, L. rhamnosus 1473 was the ly one showing an increase in total ketone amount, especially sulcatone, which is a flavoring pound found in citrus fruit [29]. Its implementation was observed by Wang, Zhang, Li, & Xu 13) [30] in grape extract, treated with β-glucosidase, but its increase has never been described after tic acid fermentation.
Overall, based on these data, L. rhamnosus 1473 was the strain that best maintained and/or reased the characteristic aromatic compounds of apple. For this reason, lacto-fermented apple mace with L. rhamnosus 1473 was chosen and used as a beer flavoring agent. Esters were one of the most representative classes observed in apple pomace fermented with L. hamnosus 1473, increasing their concentration by about seven times compared to the control. Among hem, butyl 2-methylbutanoate, hexyl acetate, hexyl n-valerate, and hexyl caproate were the most oncentrated compounds. Butyl 2-methylbutanoate a fatty acid ester with a typical apple aroma 26]increased only after L. rhamnosus 1473 fermentation and, at the same time, hexyl acetate, found in pple pomace, was preserved only during the fermentation with this strain.. Indeed, in the sample tarted with L. rhamnosus 1473, the resulting amount of hexyl acetate was statistically comparable ith the control sample; by contrast, the samples started with L. casei 2246 contained a significantly ower concentration of this compound. Hexyl n-valerate and hexyl caproate, typical apple ompounds, showed an increase when L. rhamnosus 1473 was used. Among aldehydes, benzaldehyde as the most concentrated, especially after L. rhamnosus 1473 fermentation, showing a statistically ifferent amount from the other samples (Table S1). Generally, the enzymatic conversion of henylalanine led to the formation of phenyl pyruvate, and then benzaldehyde was formed by a nonnzymatic step [27]. Concerning terpene compounds, α-farnesene, naturally occurring in apple coating [28], was the esquiterpene most concentrated (p < 0.05) in apple pomace, especially after L. rhamnosus 1473 ermentation.
Comparing all the started samples and unfermented apple pomace, L. rhamnosus 1473 was the nly one showing an increase in total ketone amount, especially sulcatone, which is a flavoring ompound found in citrus fruit [29]. Its implementation was observed by Wang, Zhang, Li, & Xu 2013) [30] in grape extract, treated with β-glucosidase, but its increase has never been described after actic acid fermentation.
Overall, based on these data, L. rhamnosus 1473 was the strain that best maintained and/or ncreased the characteristic aromatic compounds of apple. For this reason, lacto-fermented apple omace with L. rhamnosus 1473 was chosen and used as a beer flavoring agent. Esters were one of the most representative classes observed in apple pomace fermented with L. rhamnosus 1473, increasing their concentration by about seven times compared to the control. Among them, butyl 2-methylbutanoate, hexyl acetate, hexyl n-valerate, and hexyl caproate were the most concentrated compounds. Butyl 2-methylbutanoate a fatty acid ester with a typical apple aroma [26]increased only after L. rhamnosus 1473 fermentation and, at the same time, hexyl acetate, found in apple pomace, was preserved only during the fermentation with this strain.. Indeed, in the sample started with L. rhamnosus 1473, the resulting amount of hexyl acetate was statistically comparable with the control sample; by contrast, the samples started with L. casei 2246 contained a significantly lower concentration of this compound. Hexyl n-valerate and hexyl caproate, typical apple compounds, showed an increase when L. rhamnosus 1473 was used. Among aldehydes, benzaldehyde was the most concentrated, especially after L. rhamnosus 1473 fermentation, showing a statistically different amount from the other samples (Table S1). Generally, the enzymatic conversion of phenylalanine led to the formation of phenyl pyruvate, and then benzaldehyde was formed by a nonenzymatic step [27].
Comparing all the started samples and unfermented apple pomace, L. rhamnosus 1473 was the only one showing an increase in total ketone amount, especially sulcatone, which is a flavoring compound found in citrus fruit [29]. Its implementation was observed by Wang, Zhang, Li, & Xu (2013) [30] in grape extract, treated with β-glucosidase, but its increase has never been described after lactic acid fermentation.
Overall, based on these data, L. rhamnosus 1473 was the strain that best maintained and/or increased the characteristic aromatic compounds of apple. For this reason, lacto-fermented apple pomace with L. rhamnosus 1473 was chosen and used as a beer flavoring agent. Esters were one of the most representative classes observed in apple pomace fermented with L. rhamnosus 1473, increasing their concentration by about seven times compared to the control. Among them, butyl 2-methylbutanoate, hexyl acetate, hexyl n-valerate, and hexyl caproate were the most concentrated compounds. Butyl 2-methylbutanoate a fatty acid ester with a typical apple aroma [26]increased only after L. rhamnosus 1473 fermentation and, at the same time, hexyl acetate, found in apple pomace, was preserved only during the fermentation with this strain.. Indeed, in the sample started with L. rhamnosus 1473, the resulting amount of hexyl acetate was statistically comparable with the control sample; by contrast, the samples started with L. casei 2246 contained a significantly lower concentration of this compound. Hexyl n-valerate and hexyl caproate, typical apple compounds, showed an increase when L. rhamnosus 1473 was used. Among aldehydes, benzaldehyde was the most concentrated, especially after L. rhamnosus 1473 fermentation, showing a statistically different amount from the other samples (Table S1). Generally, the enzymatic conversion of phenylalanine led to the formation of phenyl pyruvate, and then benzaldehyde was formed by a nonenzymatic step [27].
Comparing all the started samples and unfermented apple pomace, L. rhamnosus 1473 was the only one showing an increase in total ketone amount, especially sulcatone, which is a flavoring compound found in citrus fruit [29]. Its implementation was observed by Wang, Zhang, Li, & Xu (2013) [30] in grape extract, treated with β-glucosidase, but its increase has never been described after lactic acid fermentation.
Overall, based on these data, L. rhamnosus 1473 was the strain that best maintained and/or increased the characteristic aromatic compounds of apple. For this reason, lacto-fermented apple pomace with L. rhamnosus 1473 was chosen and used as a beer flavoring agent. ).
Comparing all the started samples and unfermented apple pomace, L. rhamnosus 1473 was the only one showing an increase in total ketone amount, especially sulcatone, which is a flavoring compound found in citrus fruit [29]. Its implementation was observed by Wang, Zhang, Li, & Xu (2013) [30] in grape extract, treated with β-glucosidase, but its increase has never been described after lactic acid fermentation.
Overall, based on these data, L. rhamnosus 1473 was the strain that best maintained and/or increased the characteristic aromatic compounds of apple. For this reason, lacto-fermented apple pomace with L. rhamnosus 1473 was chosen and used as a beer flavoring agent.

Effect of Heating Treatment on Apple Pomace Volatiles
Both samples of apple pomace, as it is and the fermented one, were submitted to heat treatment before being used as aromatizers for beer. LAB are the predominant beer spoilers and are responsible for 60-90% of beer damage [31]. For this reason, the use of fermented apple pomace as beer flavoring agent will be possible only after a thermal treatment that could eliminate LAB. In order to evaluate the effect of the heating treatment on the volatile profile of the products, all samples were analyzed using the HS-SPME/GC-MS technique. All 45 detected volatile compounds (Table 1) were then quantified (Table S2) and data were statistically elaborated. Results are represented in Figure 2.
Generally, the heating treatment applied to apple pomace led to the formation and/or release of aromatic compounds, especially for the unstarted samples, in which the total volatile amount increased form 3.96 ± 0.14 µg/mL to 21.32 ± 0.45 µg/mL; while for the fermented apple pomace, the heating treatment did not seem to affect the already high total volatile content (21.03 ± 1.55 µg/mL and 21.77 ± 1.62 µg/mL of the unheated and heated samples respectively). As already observed for apple pomace samples, even if 2-hexenol showed a statistically significant reduction in both unstarted and fermented apple pomaces, hexanol, the main representative volatile of this class, increased after apple pomace fermentation, and was statistically augmented by the thermal treatment.
Both samples of apple pomace, as it is and the fermented one, were submitted to heat treatment before being used as aromatizers for beer. LAB are the predominant beer spoilers and are responsible for 60-90% of beer damage [31]. For this reason, the use of fermented apple pomace as beer flavoring agent will be possible only after a thermal treatment that could eliminate LAB. In order to evaluate the effect of the heating treatment on the volatile profile of the products, all samples were analyzed using the HS-SPME/GC-MS technique. All 45 detected volatile compounds (Table 1) were then quantified (Table S2) and data were statistically elaborated. Results are represented in Figure 2. Generally, the heating treatment applied to apple pomace led to the formation and/or release of aromatic compounds, especially for the unstarted samples, in which the total volatile amount increased form 3.96 ± 0.14 µ g/mL to 21.32 ± 0.45 µ g/mL; while for the fermented apple pomace, the heating treatment did not seem to affect the already high total volatile content (21.03 ± 1.55 µ g/mL and 21.77 ± 1.62 µ g/mL of the unheated and heated samples respectively).
As already observed for apple pomace samples, even if 2-hexenol showed a statistically significant reduction in both unstarted and fermented apple pomaces, hexanol, the main representative volatile of this class, increased after apple pomace fermentation, and was statistically augmented by the thermal treatment.
Esters showed a different trend: statistical analysis of the results demonstrated that their concentration decreased after the thermal treatment in the fermented apple pomace, but they were augmented in the unstarted samples. As previously reported by Kato et al. (2003) [32], the reduction of several esters, such as butyl acetate, hexyl acetate, butyl hexanoate, and hexyl hexanoate, after pasteurization of apple juice can be ascribed to a loss of these volatiles, and not to a chemical modification of the molecules.
Regarding aldehydes, the most relevant increase ascribed to the thermal treatment was noted in the unstarted apple pomace (from 1.99 ± 0.18 µ g/mL to 12.93 ± 0.01 µ g/mL), while the increase was before being used as aromatizers for beer. LAB are the predominant beer spoilers and are responsible for 60-90% of beer damage [31]. For this reason, the use of fermented apple pomace as beer flavoring agent will be possible only after a thermal treatment that could eliminate LAB. In order to evaluate the effect of the heating treatment on the volatile profile of the products, all samples were analyzed using the HS-SPME/GC-MS technique. All 45 detected volatile compounds (Table 1) were then quantified (Table S2) and data were statistically elaborated. Results are represented in Figure 2. Generally, the heating treatment applied to apple pomace led to the formation and/or release of aromatic compounds, especially for the unstarted samples, in which the total volatile amount increased form 3.96 ± 0.14 µ g/mL to 21.32 ± 0.45 µ g/mL; while for the fermented apple pomace, the heating treatment did not seem to affect the already high total volatile content (21.03 ± 1.55 µ g/mL and 21.77 ± 1.62 µ g/mL of the unheated and heated samples respectively).
As already observed for apple pomace samples, even if 2-hexenol showed a statistically significant reduction in both unstarted and fermented apple pomaces, hexanol, the main representative volatile of this class, increased after apple pomace fermentation, and was statistically augmented by the thermal treatment.
Esters showed a different trend: statistical analysis of the results demonstrated that their concentration decreased after the thermal treatment in the fermented apple pomace, but they were augmented in the unstarted samples. As previously reported by Kato et al. (2003) [32], the reduction of several esters, such as butyl acetate, hexyl acetate, butyl hexanoate, and hexyl hexanoate, after pasteurization of apple juice can be ascribed to a loss of these volatiles, and not to a chemical modification of the molecules.
Regarding aldehydes, the most relevant increase ascribed to the thermal treatment was noted in the unstarted apple pomace (from 1.99 ± 0.18 µ g/mL to 12.93 ± 0.01 µ g/mL), while the increase was ); UNST HT: unstarted heated apple pomace ( before being used as aromatizers for beer. LAB are the predominant beer spoilers and are responsible for 60-90% of beer damage [31]. For this reason, the use of fermented apple pomace as beer flavoring agent will be possible only after a thermal treatment that could eliminate LAB. In order to evaluate the effect of the heating treatment on the volatile profile of the products, all samples were analyzed using the HS-SPME/GC-MS technique. All 45 detected volatile compounds (Table 1) were then quantified (Table S2) and data were statistically elaborated. Results are represented in Figure 2. Generally, the heating treatment applied to apple pomace led to the formation and/or release of aromatic compounds, especially for the unstarted samples, in which the total volatile amount increased form 3.96 ± 0.14 µ g/mL to 21.32 ± 0.45 µ g/mL; while for the fermented apple pomace, the heating treatment did not seem to affect the already high total volatile content (21.03 ± 1.55 µ g/mL and 21.77 ± 1.62 µ g/mL of the unheated and heated samples respectively).
As already observed for apple pomace samples, even if 2-hexenol showed a statistically significant reduction in both unstarted and fermented apple pomaces, hexanol, the main representative volatile of this class, increased after apple pomace fermentation, and was statistically augmented by the thermal treatment.
Esters showed a different trend: statistical analysis of the results demonstrated that their concentration decreased after the thermal treatment in the fermented apple pomace, but they were augmented in the unstarted samples. As previously reported by Kato et al. (2003) [32], the reduction of several esters, such as butyl acetate, hexyl acetate, butyl hexanoate, and hexyl hexanoate, after pasteurization of apple juice can be ascribed to a loss of these volatiles, and not to a chemical modification of the molecules.
Regarding aldehydes, the most relevant increase ascribed to the thermal treatment was noted in the unstarted apple pomace (from 1.99 ± 0.18 µ g/mL to 12.93 ± 0.01 µ g/mL), while the increase was ); F NHT: Lactobacillus rhamnosus 1473 fermented not heated apple pomace ( before being used as aromatizers for beer. LAB are the predominant beer spoilers and are responsible for 60-90% of beer damage [31]. For this reason, the use of fermented apple pomace as beer flavoring agent will be possible only after a thermal treatment that could eliminate LAB. In order to evaluate the effect of the heating treatment on the volatile profile of the products, all samples were analyzed using the HS-SPME/GC-MS technique. All 45 detected volatile compounds (Table 1) were then quantified (Table S2) and data were statistically elaborated. Results are represented in Figure 2. Generally, the heating treatment applied to apple pomace led to the formation and/or release of aromatic compounds, especially for the unstarted samples, in which the total volatile amount increased form 3.96 ± 0.14 µ g/mL to 21.32 ± 0.45 µ g/mL; while for the fermented apple pomace, the heating treatment did not seem to affect the already high total volatile content (21.03 ± 1.55 µ g/mL and 21.77 ± 1.62 µ g/mL of the unheated and heated samples respectively).
As already observed for apple pomace samples, even if 2-hexenol showed a statistically significant reduction in both unstarted and fermented apple pomaces, hexanol, the main representative volatile of this class, increased after apple pomace fermentation, and was statistically augmented by the thermal treatment.
Esters showed a different trend: statistical analysis of the results demonstrated that their concentration decreased after the thermal treatment in the fermented apple pomace, but they were augmented in the unstarted samples. As previously reported by Kato et al. (2003) [32], the reduction of several esters, such as butyl acetate, hexyl acetate, butyl hexanoate, and hexyl hexanoate, after pasteurization of apple juice can be ascribed to a loss of these volatiles, and not to a chemical modification of the molecules.
Regarding aldehydes, the most relevant increase ascribed to the thermal treatment was noted in the unstarted apple pomace (from 1.99 ± 0.18 µ g/mL to 12.93 ± 0.01 µ g/mL), while the increase was ); F HT: Lactobacillus rhamnosus 1473 fermented heated apple pomace ( before being used as aromatizers for beer. LAB are the predominant beer spoilers and are responsible for 60-90% of beer damage [31]. For this reason, the use of fermented apple pomace as beer flavoring agent will be possible only after a thermal treatment that could eliminate LAB. In order to evaluate the effect of the heating treatment on the volatile profile of the products, all samples were analyzed using the HS-SPME/GC-MS technique. All 45 detected volatile compounds (Table 1) were then quantified (Table S2) and data were statistically elaborated. Results are represented in Figure 2. Generally, the heating treatment applied to apple pomace led to the formation and/or release of aromatic compounds, especially for the unstarted samples, in which the total volatile amount increased form 3.96 ± 0.14 µ g/mL to 21.32 ± 0.45 µ g/mL; while for the fermented apple pomace, the heating treatment did not seem to affect the already high total volatile content (21.03 ± 1.55 µ g/mL and 21.77 ± 1.62 µ g/mL of the unheated and heated samples respectively).
As already observed for apple pomace samples, even if 2-hexenol showed a statistically significant reduction in both unstarted and fermented apple pomaces, hexanol, the main representative volatile of this class, increased after apple pomace fermentation, and was statistically augmented by the thermal treatment.
Esters showed a different trend: statistical analysis of the results demonstrated that their concentration decreased after the thermal treatment in the fermented apple pomace, but they were augmented in the unstarted samples. As previously reported by Kato et al. (2003) [32], the reduction of several esters, such as butyl acetate, hexyl acetate, butyl hexanoate, and hexyl hexanoate, after pasteurization of apple juice can be ascribed to a loss of these volatiles, and not to a chemical modification of the molecules.
Regarding aldehydes, the most relevant increase ascribed to the thermal treatment was noted in the unstarted apple pomace (from 1.99 ± 0.18 µ g/mL to 12.93 ± 0.01 µ g/mL), while the increase was ).
Esters showed a different trend: statistical analysis of the results demonstrated that their concentration decreased after the thermal treatment in the fermented apple pomace, but they were augmented in the unstarted samples. As previously reported by Kato et al. (2003) [32], the reduction of several esters, such as butyl acetate, hexyl acetate, butyl hexanoate, and hexyl hexanoate, after pasteurization of apple juice can be ascribed to a loss of these volatiles, and not to a chemical modification of the molecules.
Regarding aldehydes, the most relevant increase ascribed to the thermal treatment was noted in the unstarted apple pomace (from 1.99 ± 0.18 µg/mL to 12.93 ± 0.01 µg/mL), while the increase was less considerable in the fermented apple pomace. These results are in accordance with those observed in blackcurrant juice, in which aldehyde and furan quantities were increased after thermal treatments [33]. Among aldehydes, a decrease of hexanal and 2-hexenal in fermented samples could be ascribed to enzymatic activity, as reported in apple already by Su & Wiley (1998) [34]. The concentrations of furfural and benzaldehyde were raised both in unstarted and fermented apple pomace after the thermal treatment because of Maillard reaction, as has been shown for other vegetal matrices as blackcurrant juice [33], but, in our case, the increase remained more restrained in fermented samples. Since furfural and benzaldehyde present bready, caramel, and almond notes, a high content may alter the flavor of apple pomace and generate an intense, cooked sensation. The fermented and heated sample was less abundant in these compounds, and, for this reason, it could be more appreciated as an aromatizer in beer.
The concentration of terpenes increased with the thermal treatment both in unstarted and fermented samples, and was statistically higher in the fermented one. This class of volatile is mainly characterized by the presence of α-farnesene, which increased with the thermal process. This is consistent with the results obtained by Sádecká et al. (2014) [35] in orange juice for other terpenes besides farnesene, such as d-limonene, α-pinene, etc.
Ketone concentration, mainly given by the presence of sulcatone, decreased after the thermal treatment in fermented apple pomace, even though the value resulted higher than the unfermented samples.
The heating step applied to apple pomace samples led to the formation and/or to a release of aromatic compounds in the unstarted product, and did not significantly affect the already high volatile content of the fermented samples. Differently from other studies in which a conventional pasteurization treatment has led to a loss in volatiles, in particular aldehydes such as hexanal and esters such as ethyl acetate and ethyl butyrate [36], the heating process applied in this work permitted enhancement of the flavoring compounds, especially aldehydes, alcohols, and terpenes, so as to maintain the content of aromatic compounds formed during fermentation.

Effect of Apple Pomace Addition on Volatile Profile of Beer
The volatile fractions of beer supplemented with the heated unstarted and fermented apple pomace at time zero and after 30 days of maturation was characterized by HS-SPME/GC-MS technique, and it showed a composition of 59 different aromatic compounds ( Table 2; Table S3).
As observed for apple pomace, the compounds observed in beer also belonged to five chemical classes: esters, aldehydes, ketones, alcohols, and terpenes. The most abundant class, both for number and quantity of molecules, was that of esters ( Figure 3). Our results showed that the total amount of esters was statistically comparable between the control sample and the beer aromatized with fermented apple pomace, while the beer with added unstarted apple pomace showed a lower quantity of esters. Within this class, the main representative compounds were ethyl acetate, present also in apple pomace, and ethyl octanoate, characteristic of beer volatile profile [37].  The second main class of aromatic compounds represented was that of alcohols, mostly represented by isoamyl alcohol, phenylethyl alcohol, hexanol, and isobutyl alcohol, as demonstrated in previous studies [37]. The total alcohol amount showed the same trend of esters. Some alcohols were detected in higher amounts in aromatized beer, because they were characteristic of apple: 2ethy-1-hexanol, benzyl alcohol, hexanol, isobutyl alcohol, and phenylethyl alcohol. Moreover, these compounds were statistically more concentrated in beer flavored with fermented apple pomace than in control or in beer flavored with apple pomace.  The second main class of aromatic compound represented by isoamyl alcohol, phenylethyl alcohol, h in previous studies [37]. The total alcohol amount sho were detected in higher amounts in aromatized beer, ethy-1-hexanol, benzyl alcohol, hexanol, isobutyl alcoh compounds were statistically more concentrated in bee in control or in beer flavored with apple pomace. The second main class of aromatic compounds represented was that of alcohols, mostly represented by isoamyl alcohol, phenylethyl alcohol, hexanol, and isobutyl alcohol, as demonstrated in previous studies [37]. The total alcohol amount showed the same trend of esters. Some alcohols were detected in higher amounts in aromatized beer, because they were characteristic of apple: 2-ethy-1-hexanol, benzyl alcohol, hexanol, isobutyl alcohol, and phenylethyl alcohol. Moreover, these compounds were statistically more concentrated in beer flavored with fermented apple pomace than in control or in beer flavored with apple pomace.
A similar trend was also observed for terpenes and derivatives and for aldehydes. Even if the total amount of terpenes, terpene derivatives, and aldehydes was similar between control samples and beer in which fermented apple pomace was used as an aromatizer, the concentration of α-farnesene and nonanal was more elevated in this last sample, demonstrating again the contribution of fermented apple pomace.
Ketones showed a higher concentration in beer supplemented with fermented apple pomace, and the presence of sulcatone, with characteristic citrus notes, could be ascribed to the addition of apple pomace.
Since commercial beers are subjected to a maturation time of 30 days before their commercialization, the beer samples considered in this study were analyzed at time zero and 30 days after bottling, to follow the evolution of the volatile fraction. The profiles obtained after the maturation time were comparable with those of the starting samples, in terms of volatile composition, while quantitatively, a reduction of the total aromatic compound amount was observed in all the beer samples after 30 days.
In order to underline differences and/or analogies among beer samples on the basis of the ingredient used for the aromatization after the maturation time, principal component analysis (PCA) was performed, using as independent variables the concentrations of all the detected volatile compounds (59 variables corresponding to the 59 identified compounds, listed in Table 2), extracting two components and applying a covariance matrix. The first two components explained more than the 99% of the total variance, as shown in Figure 4. PC1 was important in discrimination between beer with added heated apple pomace (Beer_UNST) and beer with added heated fermented apple pomace (Beer_F). This differentiation was possible thanks to benzaldehyde (peak 35) amount, higher in samples with the added sterilized product, and to 2-nonanone (peak 20), hexyl n-valerate (peak 25) and 2-ethyl-1-hexanol (peak 32) concentrations, more elevated in samples aromatized with fermented apple pomace. Component 2 contributed to distinguishing aromatized beers form their relative control samples (Beer_CONTR). After 30 days of maturation, control samples were indeed poor in 2-heptenal (peak 14), 2-hexenol (peak 22), and butyl caproate (peak 23), volatiles characteristic of apple pomace, but at the same time, methyl geranate (peak 45) was better maintained in these samples with respect to aromatized beers.
It is possible to conclude that the statistical unsupervised approach applied to data, PCA, represented a useful tool to determine the contributions of the addition of heated apple pomace, fermented and unstarted, to beer. samples with respect to aromatized beers.
It is possible to conclude that the statistical unsupervised approach applied to data, PCA, represented a useful tool to determine the contributions of the addition of heated apple pomace, fermented and unstarted, to beer.

Conclusions
This study investigated, for the first time, the use of lacto-fermented apple pomace as an aromatizer for beer. Results demonstrated that lactic acid bacteria can grow and modify the volatile profile of this by-product, leading to a beer characterized by distinctive aromatic features. Future studies will be focused on the exploitation of different apple varieties as substrates for fermentation. At the same time, a large number of lactic acid bacteria will be used as a starter for apple pomace fermentation in order to increase the knowledge on fermented apple pomace as a beer flavoring and to produce artisanal fruit beers which could satisfy consumer expectations.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Table S1: Influence of LAB strain fermentation on the volatile profile of apple pomace, Table S2: Influence of heating treatment and fermentation with Lactobacillus rhamnosus 1473 on the volatile profile of apple pomace, Table S3

Conclusions
This study investigated, for the first time, the use of lacto-fermented apple pomace as an aromatizer for beer. Results demonstrated that lactic acid bacteria can grow and modify the volatile profile of this by-product, leading to a beer characterized by distinctive aromatic features. Future studies will be focused on the exploitation of different apple varieties as substrates for fermentation. At the same time, a large number of lactic acid bacteria will be used as a starter for apple pomace fermentation in order to increase the knowledge on fermented apple pomace as a beer flavoring and to produce artisanal fruit beers which could satisfy consumer expectations.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/8/8/309/s1, Table S1: Influence of LAB strain fermentation on the volatile profile of apple pomace, Table S2: Influence of heating treatment and fermentation with Lactobacillus rhamnosus 1473 on the volatile profile of apple pomace, Table  S3