ITALIAN-TYPE SALAMI WITH PROPOLIS AS ANTIOXIDANT

Abstract

There is a world trend of replacing synthetic additives with natural ones. Thus, the purpose of this study was to evaluate the use of propolis, both free and microencapsulated, as a natural antioxidant in salami. For this, salamis were added with free propolis, propolis microencapsulated and other with sodium erythorbate (control). Weight loss, colour, pH, A^sub w^, lipid oxidation, microbial spoilage and sensory acceptance were examined. Products containing propolis showed similar results to the control for colour, pH, weight loss and A^sub w^. Propolis prevented the oxidation of salami during storage; however, it led to a lower sensorial acceptance. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

There is a world trend of replacing synthetic additives with natural ones. Thus, the purpose of this study was to evaluate the use of propolis, both free and microencapsulated, as a natural antioxidant in salami. For this, salamis were added with free propolis, propolis microencapsulated and other with sodium erythorbate (control). Weight loss, colour, pH, A^sub w^, lipid oxidation, microbial spoilage and sensory acceptance were examined. Products containing propolis showed similar results to the control for colour, pH, weight loss and A^sub w^. Propolis prevented the oxidation of salami during storage; however, it led to a lower sensorial acceptance.

- Keywords: antioxidant, bee glue, microencapsulation, quality, salami

(ProQuest: ... denotes formulae omitted.)

INTRODUCTION

Salami is a product made with comminuted meat and fat that is embedded and subjected to fermentation and dlying (CAMPAGNOL et cd., 2011). The lipids present in this product give it desirable sensory characteristics and nutritional and technological properties; however, they are easily oxidised, especially the linoleic (18:2, n-6) and oleic (18:1, n-9) acids (CHASCO et cd., 1993) included in this type of sausage. Oxidation degrades these compounds into secondary products, affecting the sensory attributes and nutritional value of the final product (OSAWA et al, 2005).

To control this deterioration, in addition to the synthetic antioxidants currently used by the meat industry, it is possible and desirable to use natural antioxidants. Many natural compounds in bee products have significant antioxidant activity (NAGAI et cd., 2006), including propolis.

Propolis is a complex mixture of resinous material collected by honeybees from various plant sources (GUO et al, 2011). Several studies have shown that propolis has biological properties such as antimicrobial, antifungal, antiviral and antioxidant activities (PENA, 2008). Attributed to the flavonoids and phenolic acids, most of the biological activities observed for propolis (FUNARI and FERRO, 2006).

Thus, propolis has potential to be used as a food additive because of its natural antioxidant and antimicrobial properties. However, due to its strong flavour and relative insolubility, the use of propolis in foods has been limited (NORI et cd., 2011). Microencapsulation can solve this limitation because of its ability to mitigate undesirable flavours; reduce volatility, hygroscopicity and reactivity; and increase solubility and stability in adverse conditions (FAVARO-TRINDADE et al, 2008). A study by our research group demonstrated the feasibility of microencapsulating propolis by a complex coacervation method using isolated soy protein and pectin as a coating. The process yields an alcohol-free, stable powder with antioxidant activity and antimicrobial activity against Staphylococcus aureus, and it provides controlled release in food (NORI et cd., 2011).

Thus, the objective of this work was to use both free and microencapsulated propolis in Italian-type salami to evaluate their potential as a natural additive in this type of product.

MATERIALS AND METHODS

Salami sausage formulation and processing

The formulation followed the Brazilian legislation for Italian-type salami (BRASIL, 2000). Fresh boneless pork shoulder (600 g/kg), beef rib (200 g/kg) and pork backfat (200 g/kg) were obtained from a local supermarket. The following nonmeat ingredients were added as a proportion of the total raw meat material: sodium nitrate (0.4 g/kg), sodium nitrite (0.1 g/kg), salt (25 g/kg), dextrose (7.5 g/kg), sodium tripolyphosphate (3.0 g/kg), monosodium glutamate (1.0 g/kg), white pepper (2.5 g/kg), garlic powder (2.0 g/kg), coriander (1.0 g/kg), nutmeg (0.5 g/kg), smoke aroma (0.3 g/kg) and starter culture (0.125 g/ kg) Bactoferm F-l (Chr.Hansen®, Valinhos, Sao Paulo, Brazil) containing Staphylococcus xylosus DD-34 and Pediococcus pentosaceus PCFF-1.

Salamis were divided into three treatments: treatment SE (control) with sodium eiythorbate (0.5 g/kg), a synthetic antioxidant commercially used in salami production; treatment FP with free propolis (0.14 g/kg) prepared from the dehydration of ethanolic extract of propolis in a spray dryer and dissolved in 1 g/kg of propylene glycol; and treatment MCC with microencapsulated propolis with pectin and isolated soy protein (2.29 g/kg) prepared by complex coacervation according to methods described in NORI et cd. (2011). All treatments with propolis were calculated to have the same amount of propolis solid in the final product. The propolis was prepared from green propolis, type 12, according to Brazilian classification (PARK et cd., 2002), and had its antioxidant activity and stability confirmed (NORI et cd., 2011).

The frozen beef and pork meat were cut and grounded separately using a grinder (Hobart, HB22-2 Troy, Ohio, USA) with stainless steel disks of 5 and 10 mm holes, respectively, and pork backfat was 8 mm hole. In the mixer (Beccaro, MB-25, Säo Paulo, Brazil), pork and beef were added first, followed by the pork backfat. After mixing of the raw meats, the non-meat ingredients were added, and the pre-hydrated starter culture and the propolis or sodium eiythorbate were added last. The meat batter was stuffed into collagen casings 55 mm in diameter and 250 mm in length (Viscofan Brazil, Säo Paulo, Brazil).

The salami sausages were hung from metal rods and randomly placed in a fermentation chamber for 32 days with controlled temperature and relative humidity (RH) and protected from light. Fermentation was carried out at 23°25°C, 88-90% RH for 24 h to reach a pH between 5.0-5.2. Then, the temperature and RH were reduced to 15°-17°C and 1-2% daily until reaching 75% respectively, where it remained until the 32nd day of the process when the salamis reached water activity less than 0.90. After the end of the process, the casings were removed manually and the final product was vacuum-packaged. The packages (Cryovac, Sao Paulo, Sao Paulo, Brazil) were composed of an ethylene vinyl acetate (EVA) multilayer (55 pm, 02 trans. rate <30 cm3/m2/24 h/atm). The salami was stored away from light at ambient temperature (25°C) for 90 days.

This process was repeated three times, and each time, a sample of raw material (150 g) and three sausages from each treatment were randomly selected for analysis of quality and shelf life (1, 30, 60 and 90 days of storage).

Determination of lipid oxidation

Lipid oxidation was evaluated by measuring thiobarbituric acid reactive substances (TBARS) according to the method described by BRUNA et cd. (2001). The results were expressed as mg of malonaldehyde per kg of a sample (mg MDA/ kg). To obtain these results, the water content (% water) of the samples was quantified (PREGNOLATTO and PREGNOLATTO, 1985).

Measurement of pH, water activity (AJ and instrumental colour

The pH was determined directly from samples (three measurement of each sample) using a potentiometer (Oakton pH 300 series 35618, Vernon Hills, Illinois, USA) with automatic temperature compensation and a glass penetration electrode (Digimed, Presidente Prudente, Sao Paulo, Brazil).

The water activity was measured at the temperature of 25.0°±0.3°C in salami slices approximately 3 mm thick which were placed in capsules suitable for analysis. The water activity was determined using the analyzer Aqualab CX2T (Decagon Devices Inc., Pullman, Washington, USA). This determination was performed on the raw sausage during the drying to identify the end of the process and during the storage for 90 days, with three measurements of each sample.

Colour was monitored (three measurements of each sample) using a Minolta colorimeter (Konica Minolta, Chroma Meter, 150 CR-400, Mahwah, New Jersey, USA). Measurements of L* and a* colour values (lightness and redness respectively) were carried out in 9 pieces of each treatment on the CIELab system. The colorimeter was standardized against a white tile (Y=93.7, x=0.3160 and y=0.3323), using D65 and 10° illuminant standard observer (CIE, 1978).

Weight loss

During the drying step, three salamis from each treatment were randomly selected from different locations in the drying chamber (front, right and left) were weighed daily until the end of the process. The Equations 1 and 2 were used to calculate the daily percentage of weight loss:

...(Eq. 1)

...(Eq. 2)

Where %Wx is the total weight (%) remaining on day x, where ^oWj is the weight at the first day of drying (100%); mW is the mean weight of 3 pieces of the same treatment on day x; %Wx t is the total weight (%) remaining of the pieces on day x-1 ; mWx l is the average weight of 3 pieces of the same treatment on day x-1 ; %WLx is the weight lost (%) of each treatment on day x in relation to the start of processing. Weight loss (WL%) was expressed as the percentage of the initial weight that was lost.

Microbiological analysis

Bacterial counts of coagulase-positive Staphylococcus, Salmonella spp. and thermotolerant coliform at 45°C were performed in the raw mixture and the salamis (1st day) in duplicate to evaluate the health and safety of the process as well as the effects of processing. Lactic acid bacteria were monitored to evaluate their resistance to processing and the propolis treatments employed.

For the presumptive analysis of Salmonella spp., the VIP® kit from Biocontrol for processed foods was used as indicated by the manufacturer. To count thermotolerant coliforms Petrifilm(TM) from 3M was used and coagulase-positive Staphylococcus was counted by inoculating samples onto the surface of sterile plates with Baird-Parker agar (LANCETTE and TANINI, 2001). Lactic acid bacteria were counted using MRS agar (DeMan, Rogosa, Sharpe Agar) and incubating the plates. The colonies were counted after 72 h of incubation at 37°C (GROSSO and FAVARO-TRINDADE, 2004).

Sensory analysis

An acceptance test was performed with 50 habitual salami consumers (selected according to willingness to participate) to determine whether the addition of propolis affects the sensory properties of salami. The sensory analysis was carried out in the Sensory Analysis Laboratory of Agro-industry, Food and Nutrition at Säo Paulo University in individual tasting booths under a fluorescent white light with 50 assessments per treatment in each period (1, 30, 60, and 90 days). The samples were presented in random order. The consumer acceptance test was conducted according to MEILGAARD et cd. (1999) for the evaluation of taste, aroma, appearance and overall acceptance.

The slices of 2 mm thick were presented one at a time in disposable white plastic plates coded with random three-digit numbers. A nine-point structured hedonic scale was used, ranging from "disliked extremely" (1) to "liked extremely" (9).

Statistical analysis

TBARS, L*, a*, Aw and pH analyses were evaluated according to a completely randomised design (CRD) in a 3x4 factorial array according to the model specified in Equation 3:

...(Eq. 3)

Where, Y = value observed for the variable studied (TBARS, L*, a*, Aw and pH) in repetition k [k=l (process 1), k=2 (process 2) and k=3 (process 3)] at storage dayj (j=l (1 day), j=2 (30 days), j=3 (60 days) and j=4 (90 days)] in comparison group i [i=l (SE - control), i=2 (FP - free propolis) and i=3 (MCC - microencapsulated propolis)]; y = a constant inherent in all observations (average); T = effect of the jth storage day; G, = effect of the ith comparison group; GTtj = effect of the double interaction of comparison group i with storage dayj; eljk = experimental error associated with the variable studied in repetition k at storage time j and in comparison group i, assuming NID (0, oe2).

Weight loss during the dlying process of the salami tests was evaluated following the statistical model in Equation 4:

...(Eq. 4)

Where, Y u = value observed for the weight loss variable in repetition I [1=1 (process 1), 1=2 (process 2) and 1=3 (process 3)] of material processing k (k=l to 3) at drying time j (j=l day to j=32 days) and in comparison group i [being i=l (SE - control), i=2 (FP - free propolis), i=3 (MCC - microencapsulated propolis)]; y = a constant inherent in all observations (average); G( = effect of the ith comparison group; T = effect of jth drying time; Pk = effect of kth processing where; GT = effect of the double interaction of comparison group i with drying time j; GPlk = effect of the double interaction of comparison group iwith material processing k TPjk = effect of the double interaction of drying time j with material processing k; eljkl = experimental error associated with repetition I of material processing k, drying time j and comparison group i, assuming NID (0, oe2).

For sensory analysis, which evaluated the attributes of appearance, aroma, flavour and overall quality, a mixed model was used according to the fixed effects used in the previous model, and the random effect of the consumer was included according to the recommendations of O'MAHONY (1986).

To compare groups regarding the main effect the multiple comparisons procedure of Student's t-test was used. In the case of significant interactions, the F test was used and, when necessary, regression analysis was also performed for stability studies. All tests were performed with the aid of the Statistical Analysis System® (SAS, 1995) using the PROC MIXED procedure.

RESULT AND DISCUSSION

It was possible to produce salami with either free (pure propolis powder obtained by atomizing) or microencapsulated propolis incorporated into it. However, the microencapsulated form was easily dispersed in the material, whereas the pure for clumped.

Lipid oxidation analyses

There was no significant difference between the TBARS values (approximately 0.47 mg MDA/ kg) measured in the raw mixture and those of the salamis on the first storage day. Therefore, the presence of propolis (free and microencapsulated) and sodium erythorbate was effective and/or the conditions used in processing did not promote the oxidation process.

There was also no significant difference between the treatments with respect to TBARS values throughout the storage period under the conditions employed in this study (Fig. 1). This result allowed us to infer that, with respect to lipid oxidation, both free and microencapsulated propolis performed as well as sodium erythorbate. This result proves that the microencapsulated propolis was released from the microcapsule, because it acted as effectively as an antioxidant as the other additives used.

Fig. 1 shows a significant increase (p<0.05), although very small, in TBARS values with storage time that is independent of the treatment applied. The TBARS values, even after 90 days of storage, were considerably less than values close to 1 mg MDA/kg of fermented and dry meat sausages observed in another study (MARCO et al., 2008). This result suggests that the conditions employed in the processing and storage together with the presence of erythorbate or free or microencapsulated propolis prevented lipid oxidation in the products, contributing to their preservation.

Microbiological analysis

The Italian-type salamis were within the standards of microbiological quality (BRASIL, 2001) for coliforms at 45°C, Staphylococcus co- agulase positive and Salmonella spp. (Table 1). Thus, it is observed that the sausages were produced properly in respect to good manufacturing practices for food and is suitable for use in sensory evaluation.

Considerably lower coliform counts were observed in the salami than in the raw mixture. This reduction can be attributed to the action of lactic acid bacteria, which can inhibit competing microorganisms by producing bacteriocins and by reducing the pH through the production of organic acids (HUGAS and MONFORT, 1997). Other factors are likely to have been responsible for the reduction of undesirable microbes, such as reduced Aw, the presence of nitrite and sodium nitrate and the presence of propolis that have antimicrobial properties.

The results observed for the lactic acid bacteria counts, approximately 107 CFU/g, are consistent with other studies whose counts were 106 for the raw mixture and 103 to 109 CFU/g in fermented sausages (DEL NOBILE et al., 2009, ARO ARO et al., 2010).

The sausages produced with propolis had the same lactic acid bacteria counts as those produced with erythorbate. This result suggests that the propolis used had no antimicrobial action against the starter culture, which is one more factor favouring its use as an additive in sausages.

Weight loss

Weight loss was significantly affected by the dlying time (p<0.05) (Fig. 2) in a nonlinear manner. By the 8th drying day, the salamis had already lost about 20% of their weight, reaching a total of nearly 41% at the 32nd day. This drying behaviour differs from the linear weight loss observed by GARCIA et al. (2000) in Italian-type salami, even with a weight loss of 44%, higher than observed in this experiment. No differences were observed with respect to treatment (p>0.05).

Even with the non-linear weight loss of the salami, no crust formation was observed on its surface. This result shows indirectly that the amount of water removed from the product is dependent on the temperature and RH of the ripening chamber and the processing time (GARCIA et al., 2000); thus, it appears that the procedures were conducted in a standardised manner. Other explanation is related to the increased acidification and is related to the lower pH found in the treatments, reducing the water retention capacity of the salami. Other study has reported weight loss of until 30% (G0TTERUP etaL, 2008), showing large variations between the processing of fermented and dry sausages, which may be due variables such as the formulation, the raw materials and the processing conditions.

The above results indicate that the incorporation of propolis affected neither the drying process of Italian-type salami nor the materials used as coating agents, such as pectin and isolated soy protein.

pH

The pH measurements of the sausages (Fig. 3) showed a statistically significant interaction (p<0.05) between storage time and treatment. Time 0, in this case, indicates the salami after the end of the fermentation process, when the pH was reduced to between 5.10-5.20. This decrease is related to lactic acid production by lactic acid bacteria from the starter culture.

The pH increase during the 90 days of storage, also observed by LEE et cd. (2009), may be related to the concentration of buffering proteins, the formation of ammonia and amines resulting from proteolysis by tissue enzymes or the decreasing dissociation of electrolytes in the middle (ARO ARO et cd., 2010).

Thus, it appears that the fermentation process was conducted properly and the desired pH of 5.0-5.2 was achieved at the end of fermentation. These results are consistent with commercial samples of Italian-type salami, which has a pH ranging from 4.86-5.78 (CACCIOPPOLI et cd., 2006).

The different treatments showed the same change in pH, as shown by the curves of Fig. 3, although there were points with slightly different results between treatments, as at the 75th and 90th days. However, small differences in pH between certain treatments in some areas are not significant from a technological standpoint.

Water activity

The water activity of the salami varied over time, showing a nonlinear reduction (y = 0.86 - 0.00052x + 0.0000038x2) of Aw during the 90 days of storage.

The reduction of Awfrom 0.86 on day 1 to 0.84 90 days later may be a result of free water binding to other compounds present in the material that were not identified. Similar behaviour was observed by LEE et cd. (2009) in sausages stored for up to 120 days at room temperature of 25°C.

The observed reduction in Aw between the raw mixture (mean 0.97) and the prepared salamis at time point 1 (mean 0.86) is desirable, and it is due to the drying process, wherein the water essential for the growth of microorganisms is removed from the salami.

Salamis of all the treatments met the Aw standard established by the Technical Regulation of Identity and Quality for Italian-type salami, which is a maximum of 0.90 (BRASIL, 2000). The values obtained are also consistent with those of other study (HERRERO et cd., 2007). Thus, the incorporation of free or microencapsulated propolis left the Aw behaviour of this product unaffected.

Colour characterisation

For the Italian-type sausages studied, L* decreased linearly (y = 40.90 - 0.01596x) but modestly with storage time (p<0.05); however, there was no treatment effect. L* decreased from 40.86 to 39.38 during the storage period.

From the start of processing (raw butter) to the finished product (time 1), the salamis showed a reduction in the mean value of L* from 54.32 to 40.86. This decline seems related to the process of curing, drying and ripening. In particular, the drying process contributes to this decrease by concentrating the solids in the product by dehydration.

The L* value is highly variable in Italian-type salami sold in Brazil (CAVENAGHI and OLIVEIRA, 1999), and in typical Italian-type salami, L* values of 45.70 are observed (DEL NOBILE et cd., 2009). Thus, the L* values obtained herein are consistent with those obtained for similar products. This suggests that the incorporation of neither free nor microencapsulated propolis interfered with the lightness of the salami during its shelf life.

With respect to the a* parameter, only time contributed to the statistically significant linear increase (y = 15.15 + 0.01772x) in redness. In this case, a* increased from 15.23 to 16.77. Because it is an instrumental measure, this small difference of less than two units cannot be visually perceived by humans.

The increase in a* values may be related to the presence of viable lactic acid bacteria in the salami at the end of processing. Staphylococcus xylosus, which comprises the starter culture, shows nitrate reduction activity at the optimum temperature of 30°C, which is close to the storage temperature (25°C). This may have contributed to the formation of nitrosomyoglobin by residual nitrate reduction in the salami. Furthermore, the L* reduction observed may have helped to increase the intensity of the redness. The packaging used had an oxygen barrier and, together with storage away from light, was sufficient to prevent oxidation of the meat pigments, be- cause the rate of reddening is more affected by oxygen than by light (MARCHESI et al, 2006).

The a* values obtained also showed that there was no oxidation of nitrosomyoglobin of the salami during storage, unlike other studies that have indicated a significant correlation between the parameters of lipid oxidation (which increased with respect to time) and colour in vacuum packaged sausage (ZANARDI etal, 2002).

The values obtained are lower than the 17.517.8 observed by GARCIA et al. (2000) and higher than the 7.99 observed by DEL NOBILE et al. (2009). This difference can be attributed to the raw meat used, which may have varying amounts of myoglobin, and oxidation of the red pigment.

Sensory analysis

The acceptance showed interaction between storage time and treatment with significant differences (p<0.05) in the appearance of the Italian-type salami of different treatments.

In general, the appearance of the sausage was accepted. The FP and MCC salamis received constant evaluations over the 90 days of storage in the range of "like regularly" (mean 7.0), whereas the SE salami showed a linear increase (y = 6.4 + 0.007x) in the same period from "like slightly" (mean 6.5) to "like regularly" (mean 7.2). On days 1 and 30, the salamis of treatments FP and MCC were the most accepted (p<0.05), whereas on the remaining days, no differences were observed (p>0.05) between the treatments applied.

In general, the FP and MCC salami had the most acceptable appearance over the 90 days of storage, and the SE salami was the least accepted. For the panellists, the MCC salami was characterised mainly by presenting a uniform appearance. Thus, in regard to the attribute of appearance, the addition of propolis did not impair the product's acceptance.

Regarding the attribute of aroma, the sausages studied differed only with respect to treatment (p<0.05), with no effect of storage time. The SE salamis showed a slightly higher acceptance (p<0.05; mean 7.3, in the range of "like regularly") than those from treatments FP and MCC (means of 6.6 and 6.8, respectively, in the range of "like slightly"). The more accepted aromas were described by the judges as characteristic, smooth and pleasant. Some panellists identified the odour of propolis in the FP and MCC salamis, they were unaware of the addition of this additive at the time of the evaluation, which may explain the lower acceptance of these.

However, the MCC salami was more accepted than the FP, which confirms that the encapsulation process was effective at attenuating the aroma of propolis, although incompletely so.

At no time did any consumer comment about the presence of a rancid aroma in the products, even after 90 days of storage. This confirms that the salamis remained stable with respect to lipid oxidation, confirming the results of TBARS.

The flavour of the SE salami increased linearly (y = 6.8 + 0.0087x) during the storage period of 90 days, that of the FP salami remained constant (mean 6.6), while the MCC salami's flavour declined between the 1st and 30th days but subsequently increased until the end of the 90 days of storage (y = 6.7 - 0.026x + 0.00029x2). Until the 30th day, the treatments showed no difference between them (p>0.05), and subsequently, the SE salami was more accepted for its flavour (p<0.05) than the salami of the FP and MCC treatments, which did not differ (p>0.05).

The microcapsule incorporation was not effective for softening the flavour of the product, which was comparable to that with propolis incorporated in the free form (FP). Some consumers indicated that the FP and MCC salamis were less acceptable, with a strong residual uncharacteristic flavour that was unpleasant. However, they did not indicate that the flavour was directly from propolis. This suggests that the microcapsules released the compounds responsible for the characteristic propolis flavour during the storage, which was identified in the sensory analysis.

Only an effect of treatment was observed for the overall quality attribute. The control was significantly more accepted (p<0.05) than the treatments with propolis, which did not differ from one another. However, in general, the salamis were well accepted, with means that ranged between "like regularly" (mean 7.1 for SE) to "like slightly" (mean 6.7 for the FP and MCC treatments).

The lower acceptance assigned to treatments is possibly due to the aroma and taste of propolis, which persisted in the products. However, the difference in acceptance, although significant, was relatively small and should not be the only factor to consider in choosing or rejecting this additive, because propolis is a natural additive and has several therapeutic properties that may confer functional value to the product (PENA, 2008).

The SE salamis were the most accepted for the tested attributes, except for appearance, for which the MCC treatment was more accepted.

CONCLUSION

It is possible to prepare Italian-type salami using propolis as a replacement for the synthetic antioxidant sodium erythorbate. The products have similar technological characteristics to control products (SE treatment) and slightly lower sensory acceptance with respect to aroma acceptability. The uses of microencapsulated propolis are advantageous with regard to ease of incorporation of the additive to the raw mixture and because this process will be reduce some strong sensorial characteristic of propolis. Thus, further research is needed with other microencapsulation techniques or even other microencapsulating agents to mitigate the strong aroma of propolis.

ACKNOWLEDGEMENTS

The authors thank FAPESP for financial support (Process 07/00575-2) and scholarship provided (Process 07/576660), Chr. Hansen® (Valinhos, Sao Paulo, Brazil), Ibrac® (Rio Claro, Sao Paulo, Brazil), Viscofan® (Sao Paulo, Sao Paulo, Brazil) and Cryovac® (Sao Paulo, Sao Paulo, Brazil) for materials supply.

References

REFERENCES

Aro Aro J.M., Nyam-osor P, Tsuji K., Shimada K-I., Fukushima M. and Sekikawa M. 2010. The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages. Food Chem. 119 (1): 279.

BRASIL. 2000. Aprovar os regulamentos técnicos de identidade e qualidade de copa, de jerked beef, de presunto tipo Parma, de presunto cru, de salame, de salaminho, de salame tipo alemäo, de salame tipo calabrés, de salame tipo friolano, de salame tipo napolitano, de salame tipo hamburgués, de salame tipo italiano, de salame tipo milano, de linguiça colonial e pepperoni. P. 15, Instruçâo Normativa n° 22, Diario Oficial, Brasilia.

BRASIL. 2001. Aprova o regulamento técnico sobre padróes microbiológicos para alimentos, em anexo. Resoluçâo RDC n° 12, Ministério da Saúde: Anvisa.

Bruna J.M., Ordóñez J.A., Fernández M., Herranz B. and De La Hoz L. 2001. Microbial and physico-chemical changes during the ripening of diy fermented sausages superficially inoculated with or having added an intracellular cell-free extract of Pénicillium aurantiogriseum. Meat Sei. 59(1): 87.

Caccioppoli J., Custodio F.B., Vieira S.M., Coelho J.V. and Gloria M.BA. 2006. Aminas bioativas e características físico-químicas de salames tipo italiano. Arq. Bras. Med. Vet. Zoo. 58(4): 648.

Campagnol P.C.B., Fries L.L.M., Terra N.N., Santos B.A., Furtado A.S., Toneto E.R.L. and Campos R.M.L. 2011. The influence of achyrocline satureioides ("Marcela") extract on the lipid oxidation of salami. Ciênc. Tecnol. Aliment. 31(1): 101.

Cavenaghi A.V. and Oliveira M.N. 1999. Influência de algumas características físico-químicas e sensoriais na qualidade de salame tipo italiano fabricado no Brasil. Revista Nacional da Carne. 263: 44.

Chasco J., Beriain M.J. and Bello J. 1993. A study of changes in the fat content of some varieties of diy sausage during the curing process. Meat Sei. 34(2): 191.

CIE, International Commission On Illumination. 1978. Recommendations on Uniform Color Spaces, Color Difference Equations, Psychometric Color Terms, n. 2, Supplement, Bureau Central de la CIE, Paris.

Del Nobile M.A., Conte A., Incoronato A.L., Panza O., Sevi A and Marino R. 2009. New strategies for reducing the pork back-fat content in typical Italian salami. Meat Sei. 81(1): 263.

Favaro-Trindade C.S., Pinho S.C. and Rocha G.A. 2008. Revistió: Microencapsulaçâo de ingredientes alimenticios. Braz. J. Food Technol. 11(2): 103.

Funari C.S. and Ferro V.O. 2006. Análise de própolis. Ciênc. Tecnol. Aliment. 26(1): 171.

Garcia F.T., Gagleazzi U.A. and Sobral P.J.A. 2000. Variaçâo das propriedades físicas e químicas do salame tipo Italiano durante secagem e fermentaçâo. Braz. J. Food Technol. 3: 151.

Gotterup J., Olsen K., Knöchel S., Tjener K., Stahnke L.H. and Möller J.K.S. 2008. Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities. Meat Sei. 78(4): 492.

Grosso C.R.F. and Favaro-Trindade C.S. 2004. Stability of free and immobilized Lactobacillus acidophilus and Bifidobacterium lactis in acidified milk and of immobilized B. lactis in yoghurt. Braz. J. Microbiol. 35(1-2): 151.

Guo S.J., Fu S.J., Shen Z.Q., Zhang Z.M. and Xu Q.Q. 2011. Chemical composition, biological activity and application in animal science of propolis. A review. Proceedings of Advances in Biomedical Engineering, International Conference on Agricultural and Biosystems Engineering 98-101.

Herrero A.M., Ordóñez J.A., De Avila R., Herrans B., De La Hoz L. and Carabero M.I. 2007. Breaking strength of diy fermented sausages and their correlation with texture profile analysis (TPA) and physic-chemical characteristics. Meat Sei. 77(3): 331.

Hugas M. and Monfort J.M. 1997. Bacterial starter cultures for meat fermentation. Food Chem. 59(4): 547.

Lancette G.A. and Tanini S.R. 2001. Staphylococcus aureus and Staphylococcal Enterotoxins. In: "Compendium of methods for the microbiological examination of foods". F.P. Downes and K. Ito (Ed.) p. 387-403, American Public Health Association, Washington, DC.

Lee K.T., Lee Y.K., Son S.K., Choi S.H. and Lee S.B. 2009. Changes in various quality characteristics of short-ripened salami during storage at chilled or room temperature. Kor. J. Food Sei. An. 29: 24. Abstract.

Marchesi C.M., Cichoski A.J., Zanoelo E.F. and Dariva C. 2006. Influência das condiçôes de armazenamento sobre os pigmentos cárneos e a cor do salame italiano fatiado. Ciênc. Tecnol. Aliment. 26(3): 697.

Marco A., Navarro J.L. and Flores M. 2008. The sensoiy quality of diy fermented sausages as affected by fermentation stage and curing agents. Eur. Food Res. Technol. 226(3): 449.

Meilgaard M., Civille G.V. and Carr B.T. 1999. "Sensoiy evaluation techniques". 3rd Ed. CRC-Press, Boca Raton, FL.

Nagai T., Inoue R., Kanamori N., Suzuki N. and Nagashima T. 2006. Characterization of honey from different floral sources: Its functional properties and effects of honey species on storage of meat. Food Chem. 97(2): 256.

Nori M.P., Favaro-Trindade C.S., Alencar S.M., Thomazini M., Balieiro J.C.C. and Contreras-Castillo C.J. 2011. Microencapsulation of propolis extract by complex coacervation. Food Sei. Technol. 44(2): 429.

O'Mahony, M. 1986. "Sensoiy evaluation of food: statistical methods and procedures". 1st Ed. CRC Press, Marcel Dekker, New York.

Osawa C.C., Felício P.E. and Gonçalves L.A.G. 2005. Teste de TB A aplicado a carnes e derivados: métodos tradicionais, modificados e alternativos. Quim. Nova. 28(4): 655.

Park Y.K., Alencar S.M. and Aguiar C.L. 2002. Botanical origin and chemical composition of Brazilian propolis. J. Agr. Food Chem. 50(9): 2502.

Pena R.C. 2008. Estandarización en propóleos: antecedentes químicos y biológicos. Cieñe. Inv. Agr. 35(1): 17.

Pregnolatto W. and Pregnolatto N.P. 1985. "Normas analíticas do Instituto Adolfo Lutz: métodos químicos e físicos para análise de alimentos". 3rd Ed. Instituto Adolfo Lutz, Sáo Paulo, SP.

SAS Institute, Inc. 1995. "SAS User's Guide". Statistical Analysis Systems Institute, Caiy, NC.

Zanardi E., Dorigoni V., Badiani A. and Chizzolini R. 2002. Lipid and colour stability of Milano-type sausages: effect of packing conditions. Meat Sei. 61(1): 7.

Paper received December 2,2012 Accepted April 22, 2013