Effect of Sage ( Salvia ofﬁcinalis L.) Extract on the Survival of Staphylococcus aureus in Portuguese Alheira Sausage during Maturation

: The objective of this study was to assess the effect of sage ( Salvia ofﬁcinalis L.) extract on the survival of Staphylococcus aureus in a Portuguese non-ready-to-eat meat product ( alheira sausage). Alheira batter was produced, mixed with 0.0%, 0.5%, 1.0%, or 1.5% of lyophilised sage extract, and stuffed in natural casings. Sausages were then individually inoculated with S. aureus and left to ferment/mature at 10 ◦ C/85% RH for 10 days. Sage extract was found to inactivate S. aureus ( p < 0.001) with no signiﬁcant differences between doses. At the 10th day of maturation, S. aureus decreased in 1.146 log CFU/g (SE = 0.065 log CFU/g) in alheiras mixed with 0.5–1.0% sage extract. Nonetheless, this extract retarded the growth of indigenous lactic acid bacteria during maturation. The higher the dose, the greater the effect ( p < 0.001).


Introduction
Sage (Salvia officinalis L.) is a plant of the Lamiaceae family, native to the Mediterranean, with excellent antioxidant and antimicrobial properties [1].In turn, Alheira is a non-ready-toeat sausage produced in northern Portugal, which has in the past shown low-to-moderate prevalence of Staphylococcus aureus [2].Sojic et al. [3] incorporated sage essential oil and sage extract in raw pork sausages and documented their capacity not only to retard lipid oxidation but also to be effective against aerobic microbial growth.Thus, this study was performed to evaluate the antimicrobial effect of sage extract against S. aureus in alheira sausages during the critical stage of fermentation/maturation.

Plant Material and Extraction Procedure
Dried sage aerial parts provided by Pragmático Aroma Lda.("Mais Ervas", Trás-os-Montes, Portugal) were mechanically ground and submitted to hydroethanolic extraction by dynamic maceration of 1 g plant material with 30 mL 80% ethanol (v/v) for 1 h at room temperature.The mixtures were filtrated (7-10 µm) and the ethanolic fraction was evaporated.The remaining aqueous fraction was frozen and lyophilised.

Antioxidant and Antimicrobial Activity of the Sage Extract
The antioxidant activity was measured through two in vitro assays, using previously described methodologies [4], namely, the thiobarbituric acid reactive substances (TBARS) formation inhibition assay and the oxidative haemolysis inhibition assay (OxHLIA).The extract's capacity to inhibit the formation of TBARS was assessed using porcine brain cells as oxidizable biological substrates and the results were expressed as the extract concentration (µg/mL) required to inhibit 50% of the lipid peroxidation (IC 50 ).The extract's capacity to inhibit the oxidative haemolysis was tested using sheep blood erythrocytes as ex vivo models and the extract concentration able to promote a ∆t haemolysis delay of 60 and 120 min was calculated based on the half haemolysis time (Ht 50 values) of the haemolytic curves of each extract concentration.The results were expressed as the extract concentration (µg/mL) required to keep 50% of the erythrocyte population intact for 60 and 120 min (IC 50 ).

Inoculation of S. aureus in Alheiras
Staphylococcus aureus subsp.aureus Rosenbach (ATCC 11632) kept on a fresh slant was cultivated overnight in brain-heart infusion broth (BHI) at 37 • C. On the day of inoculation, the inoculum was prepared from a second subculture in early stationary phase (~9.0 log CFU/mL) diluted in physiological water to reach 7.0 log CFU/mL.
Five batches of alheiras proxy were prepared by soaking sliced wheat white bread (22%) in hot boiled water (60%) for 20 min.After breaking down, garlic powder (1%), red pepper powder (1%), table salt (1%), and finely-shredded cooked chicken meat (10%) were added and mixed to form a well-integrated batter.Separately, virgin olive oil (5%) was heated to ~50 • C and added with 0.0, 0.5, 1.0, or 1.5% (w/w) of the lyophilised sage extract and incorporated-still warm-into the batter.The batter was mixed throughout and stuffed in pre-washed natural pig casings to produce mini-alheiras of approximately 80-90 g.The weight in g of each alheira was annotated (W).Mini-alheiras were then inoculated by individually syringing a volume of 10 W µL of the inoculum into the test units.Through this standardised procedure, each "mini-alheira" reached a S. aureus target concentration of ~5.0 log CFU/g.The fifth batch of alheira was identically produced but without S. aureus inoculation to act as negative control in order to characterise the evolution of the physicochemical properties of the alheiras during maturation, when produced at laboratory-scale.Mini-alheiras were hung in a climate-controlled chamber (10 • C, 85% RH) for fermentation/maturation to take place for 10 days.

Microbiological and Physicochemical Analyses
All analyses were conducted on days 0 (day in which the alheiras were inoculated, before maturation), 2, 4, 6, 8, and 10.For the microbiological determinations, only inoculated alheiras were analysed.For every test unit, appropriate serial dilutions were prepared by homogenising for 2 min the entire alheira content (without the casing) in 100 mL of buffered peptone water (Liofilchem, Roseto degli Abruzzi, Italy).To determine the concentration of lactic acid bacteria (LAB), 1-mL aliquot of the dilutions were pour-plated in MRS agar (Liofilchem, Italy), overlaid with 1.2% bacteriological agar (Liofilchem, Roseto degli Abruzzi, Italy), and incubated at 30 • C for 48 h.Calculations were based on confirmed bacteria [7].For the counts of S. aureus, 0.1-mL aliquot was plated on Baird-Parker agar (Liofilchem, Roseto degli Abruzzi, Italy), supplemented with egg yolk ellurite (Liofilchem, Roseto degli Abruzzi, Italy), following ISO norms [8].Typical colonies were counted after 48 h following incubation at 37 • C. For every treatment, two runs were conducted, and microbiological determinations were done in triplicate.
Physicochemical analyses were carried out for the non-inoculated alheiras and comprised the measurement of pH, water activity (a w ), and weight loss.Weight loss was also recorded in the inoculated alheira units.The pH measurement was carried out in duplicate per test unit using a pH meter (Hanna Instruments, model HI5522, Rhode Island, USA) equipped with a HI1131 glass penetration probe.To measure a w , the alheira content was transferred into the cuvette of an Aqualab meter (model 4TE Decagon, Washington, USA), and the value was recorded after measurement stabilisation.This was repeated twice per test unit.

Statistical Analysis
Results of antioxidant and antimicrobial activities of the extract are presented as mean ± standard deviation (SD) values.Statistical differences of the mean pH, a w , and weight loss of the alheiras during maturation were obtained through one-way analysis of variance (ANOVA) with time as a factor, followed by Tukey's comparison of means (α = 0.05).The effect of sage extract on S. aureus and LAB concentration was assessed by two-way analyses of variance (sage concentration and day as factors), followed by Tukey's comparisons of means between concentrations.Statistical analysis was conducted in R software (version 4.1.0,R Foundation for Statistical Computing, Vienna, Austria).

Antioxidant and Antimicrobial Activities of the Sage Extract
The results of the TBARS formation and oxidative haemolysis inhibition assays (OxH-LIA) and the MIC obtained against E. coli and S. aureus are presented in Table 1.These outcomes reinforced the high antioxidant and antimicrobial capacities already pointed out for sage extracts.In our study, the antioxidant capacity of the sage extract measured by TBARS was better than similarly-produced sage extracts from Brazil (EC 50 = 398 µg/mL), which significantly inhibited lipid oxidation in poultry pâtés during storage [9].Lower values were obtained in OxHLIA, indicating that the extract has better antioxidant capacity against free radical-induced oxidative damage of erythrocyte membranes than against the formation of malondialdehyde and other reactive substances generated in vitro from lipid peroxidation of the porcine brain cells used in the assay.Based on previous studies, the antioxidant activity of sage extracts can be attributed to the presence of phenolic compounds, mainly rosmarinic acid and derivatives of caffeic acid [10].
Table 1.Antioxidant and antimicrobial activities of the hydroethanolic extract of sage (n = 3).
The antibacterial activity assay showed that E. coli was more resistant (MIC = 1.250 mg/mL) to the sage extract than S. aureus (0.625 mg/mL) (Table 1).This was not unexpected, since Gramnegative bacteria are generally more resistant to bioactive compounds due to the hydrophilic surface of their outer membrane [1].Yet, the aforementioned Brazilian study [8] found that their extract did not inhibit S. aureus ATCC 25923 at the concentrations tested (MIC > 5 mg/mL).

Maturation Process of Lab-Scale Produced Alheiras
Table 2 compiles the variation in pH, a w , and weight loss occurring in alheiras during maturation.As suggested by the pH, the fermentation process was slow during the first two days of maturation, and steadily continued until the end of maturation.On the other hand, the a w did not change significantly from the fourth day of maturation onwards, whereas from the sixth day, alheiras no longer dehydrated significantly (see weight loss).In contrast to the results from a wide sampling of bulk alheiras sold in Portuguese fair markets [11], our lab-made alheiras compared well to the commercialised ones in terms of pH (5.49; SD = 0.034 versus 4.83; SD = 0.563) and aw (0.9734; SD = 0.0010 versus 0.9870; SD = 0.0040).For this reason, we were confident that the 80-g mini-alheiras produced in the laboratory mimicked the actual ones and could be used as proxy for the inoculation study.

Effect of the Sage Extract on S. aureus and Lactic Acid Bacteria in Alheiras
The addition of 0.50-1.50%sage extract to the alheira batter affected the kinetics of both S. aureus and lactic acid bacteria during maturation (Figure 1).When the alheiras were not formulated with sage extract (i.e., control), the inoculated concentration of S. aureus (5.0 log CFU/g) persisted throughout maturation despite acidification and dehydration, whereas the indigenous LAB proliferated more rapidly until reaching ~10.5 log CFU/g at the 10th day of maturation.From the alheiras added with sage extract, it became evident that the higher the extract concentration, the greater the effect on the populations of S. aureus and LAB (Figure 1).While sage extract produced a (desired) inactivation effect of S. aureus, it produced a (non-desired) delay in the development of LAB due to an increase in the lag phase and a decrease in the maximum concentration.ANOVA results confirmed that both day of maturation (p > 0.001) and sage concentration (p < 0.001) exerted significant effects on the two bacterial groups (Table 3).
two days of maturation, and steadily continued until the end of maturation.On the other hand, the aw did not change significantly from the fourth day of maturation onwards, whereas from the sixth day, alheiras no longer dehydrated significantly (see weight loss).In contrast to the results from a wide sampling of bulk alheiras sold in Portuguese fair markets [11], our lab-made alheiras compared well to the commercialised ones in terms of pH (5.49; SD = 0.034 versus 4.83; SD = 0.563) and aw (0.9734; SD = 0.0010 versus 0.9870; SD = 0.0040).For this reason, we were confident that the 80-g mini-alheiras produced in the laboratory mimicked the actual ones and could be used as proxy for the inoculation study.The addition of 0.50-1.50%sage extract to the alheira batter affected the kinetics of both S. aureus and lactic acid bacteria during maturation (Figure 1).When the alheiras were not formulated with sage extract (i.e., control), the inoculated concentration of S. aureus (5.0 log CFU/g) persisted throughout maturation despite acidification and dehydration, whereas the indigenous LAB proliferated more rapidly until reaching ~10.5 log CFU/g at the 10th day of maturation.From the alheiras added with sage extract, it became evident that the higher the extract concentration, the greater the effect on the populations of S. aureus and LAB (Figure 1).While sage extract produced a (desired) inactivation effect of S. aureus, it produced a (non-desired) delay in the development of LAB due to an increase in the lag phase and a decrease in the maximum concentration.ANOVA results confirmed that both day of maturation (p > 0.001) and sage concentration (p < 0.001) exerted significant effects on the two bacterial groups (Table 3).Statistically, and deducing the effect of day of maturation, all extract concentrations (0.5, 1.0, and 1.5%) significantly reduced S. aureus counts (p < 0.001) in fermenting alheira in comparison to the control treatment (0.0%).Nonetheless, as a whole, the three sage extract concentrations did not lead to significantly different reduction levels of S. aureus, as can be inferred from three non-significant p-values of the comparisons of means (Table 3).At the 10th day of maturation, sage extract added in alheira batter at a dose between 0.5-1.0%produced an average reduction of S. aureus counts of 1.146 log CFU/g Funding: The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020).The authors are also grateful to the EU PRIMA program, the Portuguese Foundation for Science and Technology (FCT) for funding the ArtiSaneFood project (PRIMA/0001/2018), and the project "BIOMA-Bioeconomy integrated solutions for the mobilization of the Agro-food market" (POCI-01-0247-FEDER-046112) by "BIOMA" Consortium, financed by the European Regional Development Fund (ERDF) through the Incentive System to Research and Technological development within the Portugal2020 Competitiveness and Internationalization Operational Program.
Data Availability Statement: Summary data can be provided upon request.

Table 2 .
Evolution of pH, water activity (a w ), and weight loss of non-inoculated mini-alheiras characterising the process of maturation at 10 • C and 85% RH (standard deviation in brackets, n = 3).

Table 2 .
Evolution of pH, water activity (aw), and weight loss of non-inoculated mini-alheiras characterising the process of maturation at 10 °C and 85% RH (standard deviation in brackets, n = 3).Effect of the Sage Extract on S. aureus and Lactic Acid Bacteria in Alheiras