Revalorization of Cava Lees to Improve the Safety of Fermented Sausages

The revalorization of food processing by-products not only reduces the environmental impact of their disposal, but also generates added economic value. Cava lees consist of inactive cells of Saccharomyces cerevisiae, and though regarded as a valueless winery by-product, they are rich in fiber and phenolic compounds. In this study, a challenge test was performed to assess the effect of cava lees and a phenolic extract (LPE) derived therefrom on the behaviour of technological microbiota (lactic acid bacteria used as a starter culture) and the foodborne pathogens Salmonella spp. and Listeria monocytogenes during the fermentation and ripening of pork sausages. Ten batches of fermented sausages were prepared with and without cava lees or the LPE, and with or without different strains of Latilactobacillus sakei (CTC494 or BAP110). The addition of cava lees reduced the pH values of the meat batter throughout the fermentation and ripening process. No growth-promoting effect on spontaneous lactic acid bacteria (LAB) or the starter culture was observed. In contrast, the presence of cava lees prevented the growth of the tested pathogens (Salmonella and L. monocytogenes), as did the starter culture, resulting in significantly lower counts compared to the control batch. In addition, the combination of cava lees with L. sakei CTC494 had a bactericidal effect on Salmonella. LPE supplementation did not affect the pH values or LAB counts but reduced the mean counts of Salmonella, which were 0.71 log10 lower than the control values at the end of the ripening. The LPE did not exert any additional effect to that of the starters applied alone. The revalorization of cava lees as a natural ingredient to improve the microbiological safety of fermented sausages is a feasible strategy that would promote a circular economy and benefit the environment.


Introduction
The food industry generates large amounts of by-products, whose disposal is costly from both an economic and environmental point of view [1]. Nowadays, there is growing interest in the revalorization of by-products rich in components such as polyphenols, proteins, fiber or lipids, which may have technological, nutritional and food safety applications [2].
The use of fiber-rich by-products as natural ingredients is being widely evaluated as an innovative reformulation strategy of fermented foods to achieve positive nutritional

Bacterial Strains
Listeria monocytogenes strains CTC1034 (serotype 4b), 12MOB045LM (genoserotype II) and Scott A (serotype 4b) and Salmonella enterica strains CTC1003 (serotype London), CTC1756 (serotype Derby) and CCUG34136 (serotype Enteritidis, Type strain) were used for the challenge test. Cultures were prepared by growing each strain independently in brain heart infusion (BHI, Beckton Dickinson, Sparks, MD, USA) at 37 • C for 7 h and subsequently sub-cultured again at the same temperature for 18 h to reach the stationary phase. The bioprotective Latilactobacillus sakei (formerly Lactobacillus sakei) CTC494, a meat isolate producing sakacin k [34], and L. sakei BAP110 grown at 30 • C for 19 h in MRS broth were used as starter cultures. All cultures were preserved frozen at −80 • C in the growth medium supplemented with 20% glycerol as the cryoprotectant until used.

Preparation of Cava Lees
Cava lees were provided by the winery Freixenet S.A. (Sant Sadurní d'Anoia, Spain). The characterization of the composition of cava lees is detailed by Aguilera-Curiel [35] and they are mainly composed of polysaccharides (72.3% in wet weight) and a lesser percentage of proteins (8.5% in wet weight). Wet lees were centrifuged at 18,000× g for Foods 2021, 10,1916 3 of 14 10 min to remove the remaining cava. The lees were subsequently frozen in an ultralow temperature freezer (−80 • C), freeze-dried (Cryodos-50, Telstar, Terrassa, Spain) and ground. Lyophilized lees (pH = 3.2 ± 0.02) were preserved in sealed tubes protected from the light and humidity.

Phenolic Extract Preparation
The cava lees phenolic extract (LPE) was obtained according to the method described by Silva et al. [24] with some modifications. Thus, 1 g of powder lees was added to 10 mL of a mixture of ethanol/water/acetic acid (80/20/0.05) and sonicated for 30 min. The supernatant was isolated by centrifugation (2500× g for 10 min at 4 • C), transferred into a flask, and the pellet was re-extracted. The collected supernatants were evaporated under vacuum on a rotatory evaporator at 37 • C. The dry residue was weighted and stored at 4 • C until used in the sausage elaboration. The total phenolic content of the extract, expressed as mg of gallic acid equivalents (GAE)/g, was determined using the method described by Vallverdú-Queralt et al. [36].

Elaboration of Inoculated Fermented Pork Sausages
Meat batter was prepared on a pilot scale under biosafety conditions by mixing minced lean pork meat and fat (8:2) ground through a 6 mm plate and inoculating it with a mixture of the three L. monocytogenes and three Salmonella strains (same amount for each strain) at a level of ca. 6 log 10 CFU/g. Subsequently, the ground meat was mixed with (in g/kg) sodium chloride (25), dextrose (7), black pepper (3), sodium ascorbate (0.5), sodium nitrite (0.15) and potassium nitrate (0.15). In the corresponding batches, 5% (w/w) of cava lees or 0.3% (w/w) of LPE was also added (corresponding to the content of phenolic compounds expected in 5% of lees). According to a previous study, 5% of cava lees was the most effective concentration for enhancing the in vitro bacterial growth [33]. In addition, this percentage of lees is also similar to those of others plant-based by-products used in some other studies [4][5][6]10]. The total amount of water of the sausage recipe was 2.6 mL/kg, including the volume used as a vehicle to add the pathogen mixture to the ground meat and the starter culture if required. In total, 10 batches were prepared for the two experiments (Table 1).
For each batch, 80 g portions of the prepared meat batter were stuffed into Tublin10 (Tub-Ex, Tass, Denmark) permeable plastic bags using vacuum packaging and were submitted to a process of fermentation and drying consisting of 2 days at 23 • C and subsequently 19 days at 15 • C.
The pH was measured with a puncture electrode 5232 (Crison Instruments S.A., Alella, Spain) and a portable pHmeter PH25 (Crison Instruments S.A., Alella, Spain) and a w with an Aqualab 3TE device (Decagon Devices, Inc. Pullman, WA, USA) at 25 • C. Analysis was performed in duplicate at selected sampling times throughout the fermentation and ripening process.

Statistical Analysis
Analysis of variance (ANOVA) and the post-hoc Tukey HSD test at a p < 0.05 significance level was done using JMP software (SAS Institute Inc, Cary, NC, USA). To determine statistical differences in bacterial counts, pH and a w of each batch during the manufacturing or storage period, one-way ANOVA was performed, using "Time" as a fixed factor.

Effect of Cava Lees Applied in Fermented Pork Sausages (Experiment 1)
Firstly, a challenge test with Salmonella spp. and L. monocytogenes was carried out in fermented sausages spontaneously fermented or inoculated with the starter culture L. sakei CTC494, both with and without the addition of 5% of lyophilized cava lees.

Characterization of Physicochemical Parameters
Sausages supplemented with 5% (w/w) of cava lees initially had lower pH values than the unsupplemented batches due to the acidity of this winery by-product (pH 3.2 ± 0.02) ( Table 2). During fermentation, the pH of sausages inoculated with the starter culture dropped significantly to values <5.3 (p < 0.05), while spontaneously fermented sausages underwent slower and slighter acidification (L1 and C1), due to the initial low levels of LAB ( Figure 1). The subsequent increase in pH values in all batches throughout the ripening process could be explained by the formation of alkaline compounds during proteolysis [38]. In all cases, the presence of lees was associated with lower pH values. The difference in pH units in spontaneously fermented sausages with and without lees (L1 and C1, respectively) ranged from 0.46 at time zero to 0.85 at the end of the ripening. Studies on the use of citrus by-products in fermented sausages also report lower pH values due to their intrinsic acidity (e.g., orange fiber by-product pH = 3.28) [8,10,39]. A synergic effect was observed when cava lees were combined with L. sakei CTC494 (L1 + CTC494), resulting in a final pH value 1.23 units lower than in the control (C1, without cava lees or starter culture).
Values of a w gradually decreased over the 21 days of ripening (Table 2) due to the sausage drying process, with no significant differences among batches (p > 0.05), neither by the inoculation of the starter culture nor by the addition of cava lees. Table 2. Values of pH and a w (mean ± standard deviation) during the fermentation and ripening of pork sausages. Batches included sausages formulated without (C1) or with (L1) cava lees, spontaneously fermented or with the addition of a starter culture (L. sakei CTC494). Values are mean ± standard deviation of triplicates. For each sampling day, significant differences between batches are indicated by different superscript letters (p < 0.05).

Batch
cava lees or starter culture). Values of aw gradually decreased over the 21 days of ripening (Table 2) due to the sausage drying process, with no significant differences among batches (p > 0.05), neither by the inoculation of the starter culture nor by the addition of cava lees. Table 2. Values of pH and aw (mean ± standard deviation) during the fermentation and ripening of pork sausages. Batches included sausages formulated without (C1) or with (L1) cava lees, spontaneously fermented or with the addition of a starter culture (L. sakei CTC494). Values are mean ± standard deviation of triplicates. For each sampling day, significant differences between batches are indicated by different superscript letters (p < 0.05).  Figure 1 shows the growth of LAB in the different batches of sausages during fermentation and ripening. The batches formulated with a starter culture (C1 + CTC494 and L1 + CTC494) exhibited the highest LAB counts throughout the process, ranging from the initial inoculated level of 5.9 log10 CFU/g to more than 9 log10 CFU/g from day 8, and  Figure 1 shows the growth of LAB in the different batches of sausages during fermentation and ripening. The batches formulated with a starter culture (C1 + CTC494 and L1 + CTC494) exhibited the highest LAB counts throughout the process, ranging from the initial inoculated level of 5.9 log 10 CFU/g to more than 9 log 10 CFU/g from day 8, and remaining stable thereafter. The implantation of the L. sakei CTC494 starter culture was confirmed by RAPD-PCR, with 100% (eight out of eight) of the isolated colonies showing the same RAPD profile as the starter culture strain at the end of the ripening process. In contrast, LAB levels in sausages produced without a starter culture (C1 and L1) were initially ca. 1.2 log 10 CFU/g and reached 8.3 log 10 CFU/g at day 8. During the subsequent ripening process, the levels remained slightly lower than in sausages with a starter culture.

Behavior of LAB during Fermentation and Ripening
With the current study design and matrix composition, the addition of cava lees did not promote LAB growth compared to the control batches throughout the manufacturing process, whether using spontaneous fermentation or L. sakei CTC494. These results are not in accordance with those previously obtained in vitro, also using L. sakei CTC494. In that study, the supplementation of the culture medium with the same amount of cava lees (5%) resulted in a significantly higher concentration of cells in different LAB strains compared to the control (without lees); in the case of L. sakei CTC494, the maximum population density was 0.8 log 10 units higher [33]. The lower amount of readily fermentable substrate in the fermented sausage formulation (i.e., 0.7% dextrose) compared to the in vitro culture media (i.e., MRS broth with 2% dextrose [33]) did not favor the use of cava lees fiber by LAB to promote their growth. A significant growth-promoting effect of other fiber-rich by-products on specific LAB strains has been demonstrated in laboratory media [40,41], whereas the addition of various by-products (from lemon, orange, tiger nut, peach or apple) in fermented sausages that also contained easily fermentable carbohydrates (e.g., glucose, dextrose, sucrose, lactose) did not improve LAB growth [3,5,8,42]. On the other hand, in the study of Yalınkılıç et al. [10], higher LAB counts were obtained in fermented sausages with 4% orange fiber compared to the control (without added fiber), although the difference in mean counts was low (0.24 log 10 units). Figure 2 shows the behavior of the pathogenic bacteria during the fermentation and ripening of the four batches of pork sausages. The sakacin k-producing strain L. sakei CTC494 was selected as a bioprotective culture able to inhibit the growth of L. monocytogenes [34]. Although sakacin has no specific inhibitory effect on Gram-negative bacteria such as Salmonella, the presence of the starter culture accelerated acidification and resulted in a lower pH, which is known to enhance the inactivation of Salmonella [43]. The presence of 5% of cava lees also had an anti-pathogenic effect, reducing the load of Salmonella and L. monocytogenes in both types of fermented sausages (Figure 2).

Impact of Cava Lees on Pathogenic Bacteria
Regarding the antimicrobial (growth inhibition) effect against Salmonella, significantly lower counts were recorded in sausages formulated with lees (L1) at all sampling times, being up to 2.7 log 10 and 0.6 log 10 lower than in C1 (p < 0.05) at day 8 and 21, respectively. It is important to highlight that the effect of cava lees on the Salmonella levels was similar to that exerted by the starter culture. Moreover, combining cava lees with L. sakei CTC494 (L1 + CTC494) enhanced the antimicrobial effect, resulting in a reduction of 3 log 10 in Salmonella during the fermentation and ripening, which was due to both bacteriostatic and strong bactericidal effects. At the end of the process (day 21), Salmonella counts were 4.3 log 10 lower than in control sausages (C1, p < 0.05).
The growth inhibitory effect of cava lees against L. monocytogenes was similar to that of bacteriocin-producing L. sakei CTC494. Compared to the control, L. monocytogenes counts were 2.3 log 10 and 2.9 log 10 lower in fermented sausages formulated with cava lees applied alone or together with L. sakei CTC494, respectively (p < 0.05).
To date, few studies have focused on the revalorization of by-products with antimicrobial effects against food-borne bacteria, especially in fermented products. A recent study revealed that a celery by-product powder produced a significant decrease in total Enterobacteriaceae counts in cooked sausages [44]. An inhibitory effect against pathogenic and opportunistic bacteria of an apple by-product added to fermented milk permeate beverages has also been recently reported [45]. Conversely, in a study on fermented sausages supplemented with a lemon by-product, higher levels of Listeria innocua (used as a surrogate of L. monocytogenes) were recorded in comparison with the unsupplemented sausages [8]. In contrast, far more studies have assessed the antimicrobial effect of food by-product extracts rich in bioactive compounds such as polyphenols [14,18,19,24,46].
In the current study, besides the growth-inhibitory effect of the starter culture L. sakei CTC494, the lower pH values achieved at the beginning of fermentation (ca. 5.2 at day 0) in sausages supplemented with cava lees (L1 and L1 + CTC494) could be another major factor responsible for the lower pathogen counts in these batches. In fact, in hurdle technology for food preservation, the pH is considered a crucial hurdle in the control of pathogenic bacteria in fermented sausages, especially in combination with a lower a w [47]. Compared to Salmonella, L. monocytogenes is more tolerant of the harsh environment usually found at the last stages of ripening, characterized by a low pH and a w [43,48,49], which could explain its lower reduction in the batches formulated with cava lees. Finally, besides the effect of pH, components of cava lees such as polyphenols and/or organic acids could also play a role in the antimicrobial activity of this by-product. It is worth highlighting that polyphenols tend to be more active against Gram-positive than Gram-negative bacteria, which can be attributed to the different bacterial cell wall structures [19,24,27,29,50]. Regarding the antimicrobial (growth inhibition) effect against Salmonella, significantly lower counts were recorded in sausages formulated with lees (L1) at all sampling times, being up to 2.7 log10 and 0.6 log10 lower than in C1 (p < 0.05) at day 8 and 21, respectively. It is important to highlight that the effect of cava lees on the Salmonella levels was similar to that exerted by the starter culture. Moreover, combining cava lees with L. sakei CTC494 (L1 + CTC494) enhanced the antimicrobial effect, resulting in a reduction of 3 log10 in Salmonella during the fermentation and ripening, which was due to both bacteriostatic and strong bactericidal effects. At the end of the process (day 21), Salmonella counts were 4.3 log10 lower than in control sausages (C1, p < 0.05).
The growth inhibitory effect of cava lees against L. monocytogenes was similar to that of bacteriocin-producing L. sakei CTC494. Compared to the control, L. monocytogenes counts were 2.3 log10 and 2.9 log10 lower in fermented sausages formulated with cava lees applied alone or together with L. sakei CTC494, respectively (p < 0.05).
To date, few studies have focused on the revalorization of by-products with antimicrobial effects against food-borne bacteria, especially in fermented products. A recent study revealed that a celery by-product powder produced a significant decrease in total

Effect of the LPE in Fermented Pork Sausages (Experiment 2)
In order to investigate whether the antimicrobial activity of cava lees could be also attributed to its phenolic fraction, a second challenge test with Salmonella and L. monocytogenes was carried out in fermented pork sausages formulated with LPE instead of cava lees. Additionally, the effect of a bacteriocinogenic (L. sakei CTC494) and a non-bacteriocinogenic (L. sakei BAP110) starter culture was evaluated.
The total phenolic content of the LPE was 152.2 ± 3.5 mg GAE/g. According to the literature, the total phenolic content of cava or wine lees differs widely, even among studies using the same extraction and determination methodology, with mean values ranging from 26 to 254 mg GAE/g [35,51,52]. Jara-Palacios et al. describe that the phenolic content in wine lees depends on the grape variety and other factors related with the vinification process [52]. The main phenolic compounds found in cava lees are caftaric acid, catechin and epicatechin, which are also the most abundant phenols in sparkling wines [35]. It has been reported that yeast cell walls possess a high capacity to adsorb phenolic compounds from wine [53][54][55][56], the contents often being greater than in other types of winery by-products (e.g., grape seeds, stems and skin) [57]. Table 3 shows the pH and a w values of the different batches of fermented sausages. Overall, supplementation with the LPE did not affect the acidity of the product at day 0. Batches inoculated with starter cultures underwent a faster acidification. The lowest pH values were reached at days 5 and 14 of ripening in sausages prepared with L. sakei CTC494 and L. sakei BAP110, respectively. A reducing effect on the pH was also observed when the starter culture was applied with the LPE. Acidification in non-inoculated fermented sausages (C2 and E2) was slower and weaker, the lowest pH value being recorded at the end of the ripening process (ca. 5.3), as usually occurs in spontaneously fermented sausages [43,58]. The rise in pH is due to proteolysis phenomena that take place at the end of the ripening period. pH values up to 6.5 have been reported in Spanish fermented sausages [59]. However, this process is highly variable and not very controllable, especially in spontaneous fermented sausages. In fact, in the current study, a great variability could be observed at this point (day 21) in batch C1 (6.26 ± 0.38) than in C2 (5.28 ± 0.17). The use of different meat raw materials in the preparation of experiments 1 and 2 could also explain these differences, even though the batches were prepared under the same conditions. No differences in a w were observed between batches during the process, as the same drying conditions were applied in each case. Table 3. Values of pH and a w (mean ± standard deviation) during the fermentation and ripening of pork sausages. Batches included sausages elaborated without (C2) or with (E2) a cava lees phenolic extract, spontaneously fermented or with the addition of a starter culture (L. sakei CTC494 or L. sakei BAP110). Values are mean ± standard deviation of triplicates. For each sampling day, significant differences between batches are indicated by different superscript letters (p < 0.05).

Batch
As shown in Figure 3, batches with a fermentation process driven by all the strains of L. sakei starter culture (C2 + CTC494, E2 + CTC494, C2 + BAP110 and E2 + BAP110) achieved values of up to 9 log 10 CFU/g of LAB at the first 2 days of ripening. These counts remained more or less stable until the end of the ripening process. The implantation of starter cultures was monitored by RAPD-PCR analysis, which showed that 100% (eight out of eight) of the isolates from the MRS plates at the end of the ripening had the same RAPD profile as the corresponding starter culture strain, thus confirming their competitiveness and dominance over the endogenous LAB. In batches without a starter culture, the initial levels of LAB were <1 log 10 CFU/g, increasing up to 6.3 and 8 log 10 CFU/g at 2 and 21 days, respectively. starter cultures was monitored by RAPD-PCR analysis, which showed that 100% (eight out of eight) of the isolates from the MRS plates at the end of the ripening had the same RAPD profile as the corresponding starter culture strain, thus confirming their competitiveness and dominance over the endogenous LAB. In batches without a starter culture, the initial levels of LAB were < 1 log10 CFU/g, increasing up to 6.3 and 8 log10 CFU/g at 2 and 21 days, respectively.  Overall, the addition of the LPE did not affect the LAB counts in any batch. In this context, LAB have been described as highly tolerant to polyphenols in the growth environment [50,60]. Our results agree with those of Wang et al. [61] and Zhang et al. [62], who found that LAB counts were unaltered by the addition of different polyphenols to meat products. Nevertheless, fermented sausages produced with a shiitake by-product extract had higher levels of LAB [16,17]. Ultimately, LAB tolerance of phenolic compounds, and their ability to metabolize them, seems to be strain-or species-specific [50,63].

Impact of the LPE on Pathogenic Bacteria
The effect of the LPE, alone or combined with L. sakei CTC494 or L. sakei BAP110, against Salmonella and L. monocytogenes is shown in Figure 4. When no starter culture was added, the addition of the LPE had very little effect on Salmonella, whose growth during the first days of fermentation was similar to that of the control (without the LPE and starter culture). However, the mean counts of the pathogen at the end of the ripening were 0.71 log 10 lower than in the control sausages. A similar effect was observed for L. monocytogenes, although in this case the final counts in the E2 batch were similar to the control sausages (C2).
As expected, L. sakei-based starter culture strains exerted a strong antimicrobial effect on Salmonella and L. monocytogenes, resulting in significantly lower pathogen counts (by 3-4 log 10 units) at the end of ripening compared to the spontaneously fermented control sausages. No additional effect was observed when the LPE was added together with the starter cultures. No strain-specific effect was observed against Salmonella, which exhibited similar behavior with both starter cultures, in contrast with L. monocytogenes, whose behavior differed. The bacteriocinogenic strain L. sakei CTC494 not only prevented the growth of L. monocytogenes but exerted a listericidal effect from the early stages of fermentation and ripening. The non-bacteriocinogenic L. sakei BAP110 reduced but did not prevent the growth of L. monocytogenes during fermentation and had an inactivation effect during ripening; at the end of the process, the count was 1 log 10 higher than in the batches containing the bacteriocinogenic L. sakei CTC494 (C2 + CTC494 and E3 + CTC494). This enhanced lethality can be related to the already reported specific antilisterial effect of L. sakei CTC494 in other food matrixes [34,64,65].
added, the addition of the LPE had very little effect on Salmonella, whose growth during the first days of fermentation was similar to that of the control (without the LPE and starter culture). However, the mean counts of the pathogen at the end of the ripening were 0.71 log10 lower than in the control sausages. A similar effect was observed for L. monocytogenes, although in this case the final counts in the E2 batch were similar to the control sausages (C2).  Polyphenols are widely reported to have an antimicrobial effect against several pathogenic bacteria, including Staphylococcus aureus, Escherichia coli, Salmonella spp. and L. monocytogenes, mostly in the context of microbial cultures [20,24,27,29,50,66]. The results obtained here indicate that the anti-Salmonella effect of whole cava lees in spontaneously fermented pork sausages may be partially attributed to the phenolic fraction of this byproduct ( Figure 4A). However, no effect of the LPE was observed against L. monocytogenes. Although most reports describe Gram-positive bacteria as far more susceptible to polyphenols than Gram-negative bacteria [20,24,28,67], this trend was not supported by the results of the current study.
The antimicrobial efficacy of polyphenol-rich extracts against pathogenic bacteria varies greatly, depending on both the phenolic structure and the bacterial species [27,63,67]. Cetin-Karaca et al. [29] assessed the antimicrobial potential of different plant phenolic compounds against three Salmonella species, reporting that (-)epicatechin, one of the main polyphenols found in cava lees, was the most effective, although with varying degrees of sensitivity according to the species. Conversely, among the range of bacterial species tested by Silva et al. [24], a winery by-product consisting of a grape polyphenol extract showed high antimicrobial activity against two food-borne strains of L. monocytogenes, but not Salmonella. It seems that the phenolic antimicrobial activity depends not only on the type of bacteria but also on the specific strain or serotype [27].
On the other hand, sausages fermented with L. sakei starter cultures exhibited a significant reduction in Salmonella and L. monocytogenes counts, regardless of the presence of the LPE. Similarly, Tremonte et al. [8] found that the addition of a polyphenol-rich lemon byproduct did not enhance the anti-Listeria effect of a bioprotective strain of Lactiplantibacillus plantarum during the ripening of fermented sausages. Considering that LAB may be able to metabolize plant-derived polyphenols, thus significantly compromising their antimicrobial potential [50,60,63,68], it may be envisaged that in the current study, the endogenous LAB and the inoculated L. sakei technological strains could have reduced the polyphenol fraction of both the cava lees and LPE.
Further studies could be designed to elucidate the role of specific components of cava lees in the antimicrobial effect observed in pork fermented sausages, which could at least be attributed to the lower pH and, most probably to acidic compounds (such as tartaric acid) and phenolic compounds (such as caftaric acid, catechin and epicatechin) determining the by-product acidity or other bacteriostatic properties. Furthermore, considering that sensory qualities are essential for consumer acceptance of food, the potential impact of cava lees on the sensorial profile of the final product should also be addressed.