Application of Clove Stem and Cinnamon Essential Oils in Dulce de Leche



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Application of Clove Stem and Cinnamon Essential Oils in Dulce de Leche
APLICAÇÃO DE ÓLEOS ESSENCIAIS DE CRAVO E CANELA EM DOCE DE LEITE

ABSTRACT

This study aims at conducting microbiological, physical, chemical and sensory analyses on Dulce de leche, four samples were developed differing for essential oil (EO) addition: C (control), T1 (Clove), T2 (Cinnamon) and T3 (Clove and Cinnamon). After being manufactured the products were stored at 25ºC and analyzed for 210 days. The samples were analyzed for physical and chemical characterization protein, lipids, lipid oxidation, pH, moisture and ashes, and for microbial characterization was Salmonella sp., Coliforms at 45°C, Staphylococcus Coagulase positive and mesophilic bacteria counts. The sensory analysis applied was the hedonic scale for the attributes of appearance, smell, taste, texture and overall acceptance. Results obtained pointed that the addition of EO (Clove and Cinnamon) does not generate alteration in the centesimal composition, texture, color, and sensory attributes of Dulce de leche. All treatments present good acceptance rate (>70%) but when comparing the samples with EO, T2 present higher acceptance (81.67%). On 210 days T2 (Cinnamom oil) presents lower (0.0409 mg malonaldehyde/g) lipid oxidation than the others treatments.



Keywords: sensory analysis, essential oils, physical and chemical analyses.
Resumo

O presente trabalho teve como objetivo realizar análises microbiológicas, físico-químicas e sensoriais de doce de leite pastoso, foram elaboradas quatro amostras, diferindo o óleo essencial adicionado, sendo elas C (Controle), T1 (óleo essencial de cravo), T2 (óleo essencial de canela) e T3 (óleo essencial de cravo e canela). Após a fabricação os doces foram armazenados a 25ºC e analisados durantes 210 dias. As amostras foram avaliadas em relação ao teor de proteínas, lipídios, oxidação lipídica, pH, umidade e cinzas. As análises microbiológicas realizadas foram Salmonella sp., Coliformes a 45°C (Coliformes fecais), Estafilococos coagulase positiva (Staphylococcus aureus) e bactérias mesófilas. A análise sensorial aplicada foi escala hedônica para os atributos aparência, aroma, sabor, textura e aceitação global. Os resultados indicam que a adição de EO (Cravo e Canela) não alterou alterações na composição centesimental, textura, cor e sensorial dos doces de leite. Todos os tratamentos apresentaram boa aceitação(IA>70%) entretanto, comparando as amostras com óleo essencial, T2 apresentou maior IA (81,67%). Em 210 dias T2 (Canela) apresentou menor oxidação lipídica (0,0409 mg malonaldehyde/g) quando comparada aos outros tratamentos.



Pavras-chave: análise sensorial, óleos essenciais, análises físico-químicas.
INTRODUCTION

Dulce de leche is obtained by cooking milk and adding sucrose also allowing the addition of safe food substances until the convenient concentration for partial caramelization. The use of modified starch is allowed and normally used in the industry; however it should not be over 0.5 g.100 mL-1. Due to the high osmotic pressure generated it may be kept at room temperature. It presents particular taste, smell, texture and color where the glossy caramel color comes from the non-enzymatic browning process known as Maillard Reaction (Demiate et al., 2001; Madrona et al., 2009).

Herbs and spices, which are composed by essential oils (EO), are used to improve the taste and flavor of food products. But in addition, they are used as antioxidant and antimicrobial agent.

Essential oils (EO) are natural substances categorized as GRAS (Generally Recognized as safe) by the Food and Drug Administration, they are widely used due to their flavoring function. From the chemical point of view EO are complex mixtures constituted by several tens of components, and this complexity makes it often difficult to explain the aforesaid activities. According Moarefian et al. (2013) lipid oxidation and microbial contamination are factors that affect food quality and shelf life. The authors observed that the extract of Cinnamomum zeylanicum contains antioxidant compounds with the ability of scavenging superoxide anions and hydroxyl radicals.

Singh et al. (2007) observed that C. zeylanicum bark essential oil (at 0.02% of concentration) acted as a good inhibitor of primary and secondary oxidation products formation in mustard oil. 

Tomaino et al. (2005) studied the antioxidant action of basil, cinnamon, cloves, nutmeg, oregano and thymus essential oils under different temperature conditions. All essential oils tested presented free radical scavenging properties, determined through spectrophotometric method based on the reduction of stable radical DPPH (2.2-difenil-1-picrilhidrazila).

According Chacón-Villalobos et al. (2013) the type of fat present in the milk, have a greater tendency to hydrolytic rancidity, a factor that can affect the quality of Dulce de leche.

Thus, the objective of this study was to realize the addition of clove stem and cinnamon essential oils in Dulce de leche and evaluate the microbiological, physical, chemical and sensory analysis.


MATERIAL AND METHODS

The present study used semi-skimmed UHT milk (Batavo®), bicarbonate (Zaeli®), corn starch (Unilever Best foods Brasil LTDA.) and sugar granulated from Alto Alegre S.A.

The essential oils were provided by the Laboratory of Natural Products of Microbiology and Imunology Department at the Biosciences Institute of Botucatu at Paulista State University "Júlio de Mesquita". Both cinnamon (Cinnamomum zeylanicum B.) and clove (Syzygium aromaticum L.) essential oils were used. The essential oils were extracted through steam-dragging distillation for essential oils production (model MA480 - Marconi) according Beraldo et al (2013). The packages used in this study were screw cap glass jars with 50 mL capacity, properly sterilized.
Dulce de leche manufacturing and yield

Dulce de leche manufacturing was handcraft and followed the traditional process proposed by Madrona et al. (2009) with modifications (EO addition). The Dulce de leche was manufactured in open concentrator an adding milk sodium bicarbonate to correct acidity. Next, the remaining ingredients were added (corn starch and sugar). The process of Dulce de leche concentration was then started (around 100ºC). After reaching the desired concentration (65ºBrix) the cooling was conducted at 25ºC.

In order to assure a Dulce de leche with all the same features, all formulations was produced in batches and next divide them adding each essential oil in the proper concentration. The storage was in glass recipient; finally both cooling and storage were carried out at room temperature (25ºC) for 210 days.

Four different types of Dulce de leche were produced, the base formula contained 77.7% milk, 21.7% sugar, 0.5% cornstarch and 0.1% sodium bicarbonate; the difference between the formulations is in concentration and type of EO used; Control: without essential oil, T1 (0.01% of Clove stem oil), T2 (0.01% of Cinnamom oil) and T3 (0.005% of Clove Stem oil + 0.005% of Cinnamom oil). The used concentrations were determined based on preliminary data (not published) from Minimal Inhibitory Concentration (MIC).



Microbiological analyses

The samples were analyzed in duplicate after being manufactured every month for 210 days in the Laboratory of Food Microbiology at the State University of Maringá regarding the presence of coagulase positive Staphylococcus according to Vanderzant & Splittstoesser (1992), for Salmonella sp., according methodology of Food and Drug Administration (Andrews & Ham, 1998), published in Bacteriological Analytical Manual, 8th issue, for Coliforms at 45°C 3M Petrifilm Plate (EC) was used; the total mesophile counting was conducted in agar (PCA, Oxoid) according to Silva et al. (1997).


Yeld, Color and instrumental texture analysis

Dulce de leche total yield was assessed through the equation 1. presented by Pinto (1979). Yield (%) = (Milk + added ingredients in kg )* 100/Production in kg (1)

All determinations were made in triplicate, after the Duce de leche manufacture. Color was evaluated through Minolta® CR400 portable colorimeter. The system applied was CIEL*a*b*, measuring the coordinates: L*, representing luminosity in a scale of 0 (black) to 100 (white); a* representing a tone scale varying from red (0 + a) to green (0 - a) and b* representing a scale of yellow (0 + b) to blue (0 - b).

The texture (hardness) analysis was carried out through the Texturometer Stable Micro Systems Texture Analyzer TAXT Plus (Texture Technologies Corp, England). The sample characteristics were: Accessory: Probe 36 mm; Mode: strength measured in compression; Option: return to the beginning; Pre-test speed: 0.5 mm s-1; Test speed: 1.0 mm s-1; Post-test speed: 2.0 mm s-1; Distance: 1 mm.


Physical and chemical analyses

The tritable acidity, moisture and ashes content, lipids concentration and total nitrogen were conducted only once right after manufacturing Dulce de leche. All analyses were carried out in triplicate. For pH, moisture, ashes and tritable acidity analyses were applied the method described by Adolf Lutz Institute (IAL, 1985). The pH was carried out for 210 days. Carbohydrates were calculated by difference (100- protein+ashes+moisture+lipids).

The lipids concentration in the Dulce de leche was quantified through the Roese-Gottlieb method modified for sugary dairy products, having the sample first diluted in the proportion of 10g to the volume of 250 mL, soon after the grease processing was conducted with a mixture of ethyl ether, ethanol and ammonium hydroxide, the separation was later conducted in a balloon and a subsequent drying in greenhouse at 105ºC to constant weight (AOAC, 2000). Total nitrogen content was determined through micro Kjeldhal method, where the values of nitrogen were multiplied by the conversion factor 6.38 (AOAC, 2000).The lipid oxidation analysis was carried out according to method by Raharjo et al. (1992).
Sensory analysis

The sensory analysis on different Dulce de leche formulations was carried out right after its production by 80 (eighty) tasters: college students and public employees both male and female, age group of 17 to 60 years old, 51 women and 29 men. The tests were carried out in the Sensory Laboratory of Food Engineering at State University of Maringá (UEM). The Dulce de leche samples were provided to tasters (randomly codified) in a portion of 25 to 30g.



For the acceptance tests a nine-point hedonic scale (1 = highly disliked it, 9 = liked it very much) was applied to evaluate the attributes of appearance, smell, taste, texture and overall acceptance, and in five-point scale for purchase intention (1 = would certainly buy it, 5 = would certainly not buy it). The Dulce de leche acceptance rate was calculated through the formulation (Eq 2):

 (2)

Where: X= sample average; N= sample highest score given by tasters.


Data analysis

The statistical analysis was evaluated through ANOVA using Statistic 7.0. When noted meaningful differences in ANOVA, the Tukey test was applied at 5% significance in order to confirm or not the difference and assess which samples differed from the other.


RESULTS AND DISCUSSION

Microbiological analysis

For all analysis periods, for all samples, counting of coliforms and mesophilic was <1x10, the Staphylococcus did not present coagulase positive and the absence of Salmonellasp in 25g of the sample at 180 storage days. Only the control sample presented high (>103) counts, of coliforms and mesophilic on 210 days.

For coagulase positive Staphylococcus regardless of the treatment applied all Dulce de leche were according to legislation patterns. It indicates that the whole process, including the stage of filling followed the good practices of manufacturing as well as the packages were properly sterilized. The results obtained for the microbiological analyses indicated that the essential oils are active antimicrobial components and they are viable to be used in this sort of product.
Yield, Color and instrumental texture analysis

The yield was of 48.28% in weight, this obtained result was higher than the one found by Madrona et al. (2009) that was 43.20% for Dulce de leche with no whey addition. Table 1 presents the instrumental results from color and texture/hardness analysis for the different Dulce de leche formulations. It is known that color is one of the most important sensory properties, if consumers perceive a strange color in a product, they will reject it.


Table 1. Results from color and texture instrumental of Dulce de leche with no EO (C), T1 (0.01% of Clove), T2 (0.01% of Cinnamom) and T3 (0.005% Clove + 0.005% Cinnamom).




Control

Treatment 1

Treatment 2

Treatment 3

L

53.83 ± 0.58a

56.30 ± 1.13a

56.30 ± 1.74a

55.63 ± 4.58a

a*

5.67 ± 0.09a

5.47 ± 0.26a

5.47 ± 0.37a

5.47 ± 0.33a

b*

16.67 ± 0.05a

18.30 ± 0.45a

18.93 ± 0.62a

18.67 ± 0.63a

Hardness (N)

1.01 ± 1.87a

0.99 ± 4.80a

0.98 ± 3.45a

0.97 ± 0.94a

*letters on the same line do not differ one from the other at 5% significance.

It is possible to observe that the results do not differ one from the other at 5% significance, which indicates that essential oils addition does not alter the color of Dulce de leche. Bellarde (2006) instrumentally analyzed Dulce de leche color and obtained L 50.77, for a* 7.45, and for b* 24.21. When comparing Dulce de leche with essential oils addition and the presents study, it can be noted that L is slightly higher, a* is lower and b* is lower, which may be explained by the sort of equipment used, time and cooking temperature.

In the other hand Ranalli et al. (2012) evaluated two commercial samples of traditional dulce de leche and found values for L (36.4 and 44.4), a* (6.56 and 8.15) and b* (16.9 and 25.4).

We note that there was no meaningful difference between the results from hardness at 5% significance, once the Dulce de leche formulation was the same for all treatments and the variation was the addition of essential oils after the product was ready in very small quantity. Chacón-Villalobos et al. (2013) produced a dulce de leche with different goat milk concentrations (0, 25, 50, 75 and 100%) and found values around 0.9 to 5.5 N for hardness.



Physical and chemical analyses

Moisture, ashes and tritable acidity, carbohydrates, lipid oxidation and hydrogenionic potential (pH) results are presented in Table 2. There was no meaningful difference for the malonaldehyde content present in the samples on 0 days. On 210 days the Treatment 2 (Cinnamom oil addition) presents lower (0.0409 mg malonaldehyde/g) lipid oxidation than the others treatments.


Table 2. Results from the analyses of moisture, ashes, tritable acidity, carbohydrates, proteins, lipid oxidation, pH and lipids.




Control

Treatment 1

Treatment 2

Treatment 3

Moisture (%)

28.65± 0.07a

27.62 ± 0.83a

26.79 ± 0.90a

27.91 ± 0.03a

Ashes (%)

1.83 ± 0.01a

1.87 ± 0.08a

1.82 ± 0.12a

1.83 ± 0.04a

Tritable acidity (%)

0.24 ± 0.01a

0.26 ± 0.02a

0.25 ± 0.03a

0.25 ± 0.02a

Protein (%)

6.08 ± 0.17a

6.06 ± 0.06a

6.00 ± 0.00a

6.06 ± 0.06a

Lipids (%)

2.67 ± 0.00a

2.33 ± 0.06a

2.50 ± 0.00a

2.60 ± 0.00a

Carbohydrates (%)

60.77

62.12

62.89

61.60

pH (day 0)

6.81

6.80

6.80

6.79

pH (day 210)

6.80

6.79

6.80

6.80

Lipid oxidation (mg malonaldehyde/g) (day 0)

0.0339±0.05Aa

0.0330±0.004Aa

0.0261±0.002Aa

0.0263±0.004Aa

Lipid oxidation (mg malonaldehyde/g) (day 210)

0.0468±0.00Ba

0.0432±0.03Ba

0.0409±0.004Bb

0.0443±0.00Ba

*Equal letters on the same line do not differ one from the other at 5% significance.

**Capital letters on the same column do not differ one from the other at 5% significance.

The authors also observed that in general there was a moderate increase in TBA values throughout the storage time. The rancidity in dairy products can cause a perception of bitter taste caused by the presence of mono-or diglycerides. It can be consider the form the samples were storage in small glass jars with little air left inside, which contributed to the occurrence of lipid oxidation.

For pH analyses regardless the treatment applied all Dulce de leche were found to be very close to the ones found by Klein et al. (2010) 6.6, and higher than the ones found by Ferramondo et al. (1984), that ranged from 5.6 to 6.3.

It is possible to verify that there was no meaningful difference at 5% significance between formulations for none of the analyses of moisture, ashes, tritable acidity, lipids and protein, which reveals that the addition of essential oils does not generate alteration in the centesimal composition of Dulce de leche.

According Barbosa et al. (2013) from a physicochemical point of view, dulce de leche is a mixed aqueous dispersion composed mainly of sucrose and lactose (carbohydrates) and milk proteins, in which the continuous phase contains different types of dispersed particles. The typical composition of this product is 30% of moisture (maximum); 6% of proteins (minimum); 2% of lipids (minimum); 2% of ashes (minimum); and 70% of sugars (maximum) our results are similar to those cited.

The chemical composition of several Dulce de leche was assessed by Demiate et al. (2001), the authors observed that the average content was of 26.80% for moisture, 1.41% for ashes, 3.90% for lipids and 7.07% for proteins, data that if compared with the proposed study present few variations which may be due to technology applied when manufacturing commercial Dulce de leche.

According to Santos et al. (2010), the tritable acidity for Dulce de leche sold in informal businesses in Currais Novos, Rio Grande do Norte varied from 0.22% to 0.28%. The results found by the present study are approximate to the data found by such authors.
Sensory analysis

Table 3 reveals that there was no meaningful difference between the samples for none of the attributes at 5% significance. At this point it is important to highlight that the sensory analysis was carried out with non-trained tasters who liked or disliked clove and cinnamon.


Table 3. Results from sensory analysis.

Attributes

Control

Treatment 1

Treatment 2

Treatment 3

Appearance

7.79a

7.58a

7.79a

7.00a

Smell

7.75a

6.93a

7.30a

7.15a

Taste

8.06a

6.98a

7.11a

7.15a

Texture

7.60a

7.29a

7.43a

7.60a

Overall acceptance

7.83a

7.18a

7.35a

7.04a

Purchase intention

1.85a

2.59a

2.46a

2.60a

Acceptance rate

87.0%

79.78%

81.67%

78.22%

*Equal letters on the same line do not differ one from another at 5% significance.
For a product to be well accepted the acceptance level should be higher than 70%, thus, all concentrations were proven to be well accepted. The control sample was the one with the highest acceptance rate having no addition of essential oils, followed by the treatment with addition of cinnamon essential oil (T2), the one both cinnamon and clove stem essential oils (T3) and, finally, the sample with the lowest sensory acceptance rate was the one with addition of clove stem essential oil (T1).

Madrona et al. (2009) carried out sensory analysis with different concentrations of whey replacing milk and compared it with traditional Dulce de leche (with no whey adddition), and obtained average of 6.66 for overall acceptance of Dulce de leche without whey addition. Dulce de leche with addition of essential oils obtained 7.83 for control, 7.18 for treatment 1, 7.35 for treatment 2 and 7.04 for treatment 3. When establishing a comparison between the results obtained by those authors and the ones obtained by the present study the Dulce de leche with addition of essential oils presented overall acceptance similar to traditional one, which proves it to be marketable.


CONCLUSION

Dulce de leche with addition of clove stem and cinnamon essential oils or both, not presented alteration in the centesimal composition, texture, color, and microbial counts comparing with control sample for 210 days of storage. Treatment 2 (Cinnamom oil) presents lower (0.0409 mg malonaldehyde/g) lipid oxidation than the others treatments on 210 days.

All Dulce de leche samples was sensory well accepted (acceptance rate>70%). Therefore, we may state that further studies are yet to be conducted for longer periods in order to improve the development of such product, but it is currently viable to be produced in industrial scale, reaching new consumers.
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