Fernandez-Lopez, 2005.pdf

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Antioxidant and antibacterial activities of naturalextracts: application in beef meatballs

J. Fernandez-Lopez a,*, N. Zhi b, L. Aleson-Carbonell a, J.A. Perez-Alvarez a, V. Kuri b

a Departamento de Tecnologa Agroalimentaria, Escuela Politecnica Superior de Orihuela (EPSO), Universidad Miguel Hernandez, Ctra. a

Beniel Km 3,2 (03312) Orihuela, Alicante, Spainb Agri Food Centre/SBC, Seale-Hayne, University of Plymouth, Newton Abbot, UK

Received 9 February 2004; received in revised form 6 August 2004; accepted 16 August 2004

Abstract

The antioxidant and antibacterial effect of rosemary, orange and lemon extracts was investigated in cooked Swedish-style meat-

balls. Activity in a lard system was established for all the extracts and further determination of the development of rancidity as thio-

barbituric acid reactive substances consistently showed that about 50% of the rancidity can be controlled by the citrus preparations.

Two of the rosemary extracts (water soluble and oil soluble) were more effective with practically complete elimination of rancidity

(TBA values) after a period of 12 days. Rosemary extract activity against lactic acid bacteria and Listeriabut not Brochothrix ther-

mosphactawas demonstrated in an agar diffusion test, but in the product only lactic acid bacteria counts were slightly reduced. Sen-

sory analysis results, particularly aroma and acceptability scores, indicated the significant advantages in using rosemary and citrus

extracts in rancidity-susceptible meat products.

2004 Elsevier Ltd. All rights reserved.

Keywords: Rosemary; Garlic; Antioxidant; Antimicrobial; Citrus; Spoilage; Lactic acid bacteria

1. Introduction

The appearance of foods is one of the major determi-

nants of its appeal to consumers and consequently, sales

of the product. Lipid oxidation and bacterial contami-

nation are the main factors that determine food quality

loss and shelf-life reduction. Therefore, delaying lipid

oxidation and preventing bacterial cross-contamination

are highly relevant to food processors. The growth ofmicroorganisms in meat products may cause spoilage

or foodborne diseases. Oxidative processes in meat lead

to the degradation of lipids and proteins which, in turn,

contribute to the deterioration in flavour, texture and

colour of displayed meat products (Decker, Chan, Livi-

say, Butterfield, & Faustman, 1995). Several authors

have postulated that in meat and meat products pigment

and lipid oxidation are interrelated (Anton, Salgues, &

Renerre, 1993; Yin & Faustman, 1993). Genot, Borel,

Metro, Gandemer, and Renerre (1991) concluded that

O2 can initiate lipid peroxidation, leading to the forma-

tion of prooxidant substances capable of reacting with

oxymyoglobin (OMb) and resulting in metmyogobin

(MMb) formation. Anton et al. (1993) postulated that

OMb could be oxidized not only by lipid-oxy radicalsbut by other pro-oxidant radicals generated by O2. Sev-

eral investigators reported that the susceptibility of

myoglobin to autoxidation is the main factor in explain-

ing colour stability in meat and meat products (Renerre,

Anton, & Gatellier, 1992).

Although synthetic additives have been widely used

in the meat industry to inhibit both, the process of lipid

oxidation and microbial growth, the trend is to decrease

their use because of the growing concern among

0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.meatsci.2004.08.004

* Corresponding author. Tel.: +34 966749656; fax: +34 966749677.

E-mail address: [email protected](J. Fernandez-Lopez).

www.elsevier.com/locate/meatsci

Meat Science 69 (2005) 371380

MEATSCIENCE
mailto:[email protected]:[email protected]:[email protected]


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consumers about such chemical additives (Chastain,

Hufman, Hsieh, & Cordray, 1982; Chen, Pearson, Gray,

Fooladi, & Ku, 1984). Consequently, search for natural

additives, especially of plant origin, has notably in-

creased in recent years (Loliger, 1991). Compounds ob-

tained from natural sources such as grains, oilseeds,

spices, fruit and vegetables have been investigated(Chen, Muramoto, Yamauchi, & Huang, 1996). There-

fore, the development and application of natural prod-

ucts with both antioxidants and antibacterial activities

in meat products may be necessary and useful to pro-

long their storage shelf life and potential for preventing

food diseases.

Rosemary (Rosmarinus officinalis L.) has been

reported to contain certain compounds including, rosm-

anol, rosmariquinone, rosmaridiphenol and carnosol,

which may be up to four times as effective as butylated

hydroxy anisole (BHA) and equal to butylated hydroxy

toluene (BHT) as antioxidants (Houlihan, Ho, &

Chang, 1984, 1985; Nakatani & Inatani, 1984). Moreo-

ver, several authors reported that some compounds pre-

sent in rosemary extracts could have antibacterial

activity (Cuvelier, Richard, & Berset, 1996).Del Campo,

Amiot, and Nguyen-The (2000) reported that the com-

pounds responsible for the antibacterial action seemed

presumably to be the phenolic di-terpenoids, which are

the main compounds of the apolar fraction of the rose-

mary extracts.

Garlic extracts have also been shown to have antioxi-

dant activity in different in vitro models. The antioxi-

dant activity of Allium plants has mainly been

attributed to a variety of sulphur-containing compoundsand their precursors (Kim, Kubota, & Kobayashi, 1997;

Lampe, 1999; Nuutila, Puupponen-Pimia, Aarni, &

Oksman-Caldentey, 2003). These compounds have been

also reported as responsible for their in vitro antibacte-

rial activity (Harris, Cottrell, & Plummer, 2001; Tsao &

Yin, 2001).

Citrus fruits are an important source of bioactive

compounds (flavonoids and vitamin C). The main flav-

onoids found in citrus species are hesperidine, narirutin,

naringin and eriocitrin (Mouly, Arzouyan, Gaydou, &

Estienne, 1994; Schieber, Stintzing, & Carle, 2001).

Ascorbic acid, a well known natural antioxidant, to-

gether with natural flavonoids are also attracting more

and more attention not only due to their antioxidant

properties, but as anti-carcinogenic and anti-inflamma-

tory agents because of their lipid anti-peroxidation ef-

fects (Elangovan, Sekar, & Govindasamy, 1994;

Martn, Frutos, Perez-Alvarez, Martnez-Sanchez, &

Del Ro, 2002).

As can be seen, several studies have demonstrated

the antibacterial and/or antioxidant properties of these

plants, mainly using in vitro assays. Moreover, some

researchers reported that there is a relationship be-

tween the chemical structures of the most abundant

compounds in the plants and their above mentioned

functional properties (Dean & Svoboda, 1989; Farag,

Daw, Hewedi, & El-Baroty, 1989). Because these com-

pounds are likely to interact with their environment,

and the composition could vary according to regional

conditions, the investigation of their activity in a range

of food systems is still needed to successfully applythem into meat products. The objectives of this study

were to investigate (I) the antioxidant activities of com-

mercially available natural extracts, (II) the ability of

these natural extracts for inhibiting the growth of 11

foodborne bacterial contaminants and (III) their ability

to extend the storage shelf life of a cooked meat

product.

2. Materials and methods

2.1. Natural extracts

Rosemary and garlic extracts: Three different com-

mercial rosemary extracts and one of garlic were pro-

vided by Kalsec (Kalsec Mildenhall, UK): Herbalox

seasoning Type HT-O which is an rosemary oil miscible

extract, Duralox oxidation management Blend CN-2

which is a rosemary water miscible extract, Herbalox

seasoning Type W which is a rosemary oil and water

miscible extract, and Aquaresin garlic which is a garlic

water miscible extract.

Citrus extracts (orange and lemon): these extracts

were obtained from by-products of lemon and orange

juice industries. As these by-products have a high watercontent (80%) which would make their application in

the food industry difficult, it was necessary to process

them to obtain a dry powder of aprox. 7% moisture.

The process described by Fernandez-Gines, Fernan-

dez-Lopez, Sayas-Barbera, Sendra, and Perez-Alvarez

(2003) included some built-in precautions to minimize

the losses of the associated bioactive compounds; flavo-

noids and ascorbic acid.

2.2. Bacterial cultures

The eleven foodborne bacteria used as test organisms

were selected as follows: Listeria innocua 4202, Listeria

monocytogenes 5105, Lactobacillus sake 550, Leuconos-

toc mesenteroidessubsp. mesenteroides824, Leuconostoc

mesenteroides subsp. dextranicum 882, Leuconostoc car-

nosum 558, Lactobacillus curvatus 860 Brochothrix ther-

mosphactaCRA 7883, Brochothrix thermosphacta CRA

7884,Brochothrix thermosphactaCRA 3235, and Lacto-

coccus lactis FMRD 492. All isolates from vacuum

packed spoiled meat products (Borch & Molin, 1988;

Davies et al., 1999) except the last one (FMRD 492).

FMRD strain was obtained from the Agricultural and

Food Research Centre (DARDNI, Belfast, UK) and

372 J. Fernandez-Lopez et al. / Meat Science 69 (2005) 371380


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CRA strains are from Campden and Chorleywood

(Food Research Association, Chipping Campden,

UK). All of them were selected as markers for meat

products deterioration.

2.3. Determination of antioxidant effect in natural

extracts (Rancimat method)

The American Oil ChemistsSociety (AOCS) air oxi-

dation method (AOM-AOCS Cd 12b-92) was used to

determine the antioxidant effect of each extract (Laubli

& Bruttel, 1986). All experiments were performed with

a 679 Rancimat (Metrohm, Herisau, Switzerland). Each

extract (at the percentages showed inTable 1) was added

to 2.5 g of melted commercial pork lard (Co-op, UK)

into the reaction vessel. Then, they were inserted into

the heating block for 10 min to preheat the sample

(120 C). The air supply and the absorption vessels were

connected and recording of the conductivity curves

started. Lard (without extract added) was used as con-

trol sample. The results are expressed as stability index

(SI), calculated as a ratio of the induction time of the

treatment sample and the induction time of the control.

Three replications of this experiment were made.

2.4. Determination of antibacterial effect in natural

extracts

Antimicrobial activity of natural extracts against 11

bacteria was tested using the agar diffusion method

(Daeschel, 1992; Kuri, 1998). Stock cultures of all tested

bacteria were grown in nutrient broth (Oxoid UnipathLtd., Basingtoke, UK) for 18 h. Final cell concentra-

tions were standardized until 107108 cfu/ml using the

McFarland solutions standards (NCCLS, 1990). One

millilitre of this inoculum was added to each plate con-

taining soft brain heart infusion (BHI) agar (Oxoid Uni-

path Ltd., Basingtoke, UK). When the agar was

solidified, 4 wells (6 mm diameter) were formed in each

plate. Fifty microlitre of each extract was applied into

each well. The control sample was prepared with the

same amount of sterile distilled water instead of extract.

After a diffusion time of 1215 min at room tempera-

ture, the plates inoculated with Brochothrix spp. were

incubated at 22 C for 48 h. All lactic acid bacteria

(LAB) were incubated at 30 C for 48 h and Listeria

spp. at 37 C for 48 h. After this time, the inhibition

diameter was measured in 3 directions and the average

tabulated or used to calculate activity units. Three repli-

cations of this experiment were made.

2.5. Meatball manufacture

2.5.1. Product formulation

Swedish-style meatballs were manufactured accord-

ing to a conventional formula: 78% minced beef (20%

fat content), 14.5% flake potatoes, 5% water and 2.5%

salt. Sunflower oil (1.5%) was used as a carrier for the

extracts. A set of 6 treatment samples differing only by

the extract added were prepared (Table 1).

Garlic extract was not used in meatballs manufacture

due to their pro-oxidant activity showed with rancimat

method and to their strong aroma showed in previous

assays.

Rosemary extracts were used at the concentrations

recommended by the supplier for its use in meat prod-

uct. Citrus extracts were used at the concentration sug-

gested by previous studies (Fernandez-Gines et al.,

2003; Fernandez-Lopez et al., 2004).

2.5.2. Product processing

The products were prepared in a pilot plant resem-

bling to commercial processing conditions. All ingredi-

ents were homogenized in a bowl mixer with a spiral

dough hook (Silverson, Birmingham, UK) during

5 min. For each treatment, the corresponding extractwas added at the concentrations shown in Table 1,

and then mixed again for 5 min. Meatballs were formed

by hand (15 g, 2025 mm) and then subjected to a two-

stage cooking process. First the meatballs were flash

fried into sunflower oil at 190 C for 30 s. The objective

of this stage was to seal the surface of the ball and pro-

duce the characteristic browned look. They were then

thoroughly cooked in a forced draught oven (Zanussi,

Italy) at 250 C during 4 min to reach an internal tem-

perature of 72 C in the center of the meatball. The tem-

perature was monitored using an Omega digital

thermometer (Omega Engineering, Inc., Stamford, CT)

with a chromelalumel (Omega K) thermocouple probe

positioned in the geometric center of the product sam-

ples. When the endpoint temperature was achieved,

the samples were immediately placed in a chiller (2

5 C) to reach a product temperature below 12 C. Three

replications of this experiment were made.

2.5.3. Storage conditions

After reaching the packing temperature the samples

were placed into plastic containers, sealed with one layer

of a semi-permeable film (Polyvinylidene chloride,

Freshcling, Plymouth, UK), and stored in darkness at

Table 1

Type and amount of each natural extract used in this study

Treatment Extract Percentage

(%, w/w)

Control

Rosemary oil extract (OR) Herbalox Type HTO 0.10

Rosemary water extract (WR) Duralox 0.15

Rosemary oil and

water extract (OWR)

Herbalox Type W 0.25

Garlic extract (GR) Aquaresin garlic 0.05

Lemon fiber (LF) Lemon extract 5.00

Orange fiber (OF) Orange extract 5.00

J. Fernandez-Lopez et al. / Meat Science 69 (2005) 371380 373


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8 1 C for 12 days to follow an accelerated shelf life

determination protocol (IFST, 1993). Sampling and

storage conditions records from each treatment took

place at 1, 3, 6, 9 and 12 days (storage time) and every

sample was analysed promptly as follows.

2.6. Meatballs analysis

2.6.1. Colour measurements

Meat colour was measured using a Minolta colorime-

ter CR-200 (Minolta Camera Co., Osaka, Japan) with

illuminant D65, 2observer, Diffuse/O mode, 8 mm aper-

ture of the instrument for illumination and 8 mm for

measurement. The colorimeter was standardized with a

white tile (L* = 98.14, a* = 0.23 and b* = 1.89). Colour

was described as coordinates: lightness (L*), redness (a*,

red-green) and yellowness (b*, yellow-blue). Nine rep-

licate measurements were taken for each sample, follow-

ing the guidelines for colour measurements from

American Meat Science Association (Hunt et al., 1991).

2.6.2. TBA values

Lipid oxidation was assessed in triplicate by the

2-thiobarbituric acid (TBA) method of Tarladgis,

Watts, and Younathan (1960)with minor modifications.

A 10 g sample was blended with 50 ml distilled water for

2 min and then transferred to a distillation tube. The cup

used for blending was washed with an additional 47.5 ml

distilled water, which was added to the same distillation

tube with 2.5 ml 4N HCl and a few drops of antifoam

agent silicone o/w (Fisher Scientific, Loughborough,

UK). The mixture was distilled and 50 ml distillatewas collected. Five ml of 0.02 M 2-thiobarbituric acid

in 90% acetic acid (TBA reagent) was added to a vial

containing 5 ml of the distillate and mixed well. The

vials were capped and heated in a boiling water bath

for 30 min to develop the chromogen and cooled to

room temperature. The absorbance was measured at

538 nm, against a blank prepared with 5 ml distilled

water and 5 ml TBA-reagent, using a PV 8625 spectro-

photometer (Philips, UK). Thiobarbituric acid-reactive

substances (TBARS) were calculated from a standard

curve (850 nmol) of malondialdehyde (MA), freshly

prepared by acidification of TEP (1,1,3,3-tetraethoxy

propane). Reagents were obtained from Sigma (UK).

The TBA numbers were calculated as mg MA/kg

sample.

2.6.3. Microbiological analysis

A composite sample (10 g) was formed with portions

of at least 3 meatballs and homogeneized with sterile

1.5% peptone water, in a Stomacher 400 (Colworth,

London, UK) for 1 min. Aliquotes were serial diluted

in peptone water and plated out following standard

methodologies (Gerhardt et al., 1994). Lactic acid bacte-

ria (LAB) counts were determined on MRS Agar (pH

5.6), with the plates incubated under anaerobic condi-

tions (Gas generating kit anaerobic system, Oxoid Uni-

path Ltd., Basingtoke, Hampshire, UK) at 30 C for

2 days. Psychrotrophic microbial counts were deter-

mined on Plate Count Agar (PCA) with plates incubated

at 7 C for 10 days. Coliform organisms were enumer-

ated on MacConkey Agar (MCA) plates incubated at30 C for 48 h. Moulds and yeasts were determined on

RBC (Rose bengal cloramphenicol) agar plates incu-

bated at 22 C for 5 days. Culture media were from Ox-

oid (Oxoid Unipath Ltd., Basingtoke, Hampshire, UK).

Results were expressed as log10cfu/ml.

2.6.4. Sensory evaluation

Sensory evaluation followed standard guidelines ISO

6658-1995 and ISO 6564-1985 (British Standards, 1986a,

1986b). A preliminary descriptive evaluation was per-

formed on commercial Swedish meatballs (Crown

Brands International, Ltd, Swindon, UK) and samples

made as described above to identify and define principal

sensory attributes and find consensus. Each meatball

sample was evaluated by a trained 4-member panel.

The same panel evaluated samples at each storage time

(1, 6, 9 and 13 days storage). The sensory questionnaires

measured intensity on a 7-point balanced semantic scale

(weak to strong) for the following attributes colour (cro-

ma), brightness, surface-slime, aroma (rancidity), aroma

(putrefaction), aroma (off odour), aroma (acid), aroma

(sour) and overall acceptance.

2.7. Statistical analysis

Conventional statistical methods were used to calcu-

late means and standard deviations. Statistical analysis

(ANOVA) was applied to determine significant differ-

ences (P< 0.05). To discover where there were significant

differences between the levels of the main factor, contrasts

(Tukey test) between means were made (Afifi & Azen,

1979). For the antioxidant and antimicrobial properties

of natural extracts ANOVA withtwo factors (extract with

seven levels: control, OR, WR, OWR, GR, OF and LF,

and replication with three levels) were applied. For the

meatball analysis, ANOVA with three factors (treatment

with six levels: control, OR, WR, OWR, OF and LF, stor-

age time with four levels: 1, 6, 9 and 12 days, and replica-

tion with three levels) were applied for each parameter.

Statgraphics Plus 5.1 for Windows (Statgraphics Co.,

Tulsa, OK, USA) was used for statistical analysis.

3. Results and discussions

3.1. Antioxidant activity of natural extracts

Antioxidant activities of each natural extract ex-

pressed as stability index (SI) are shown in Table 2.



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The only extract that did not show any antioxidant

properties was garlic extract (GR), which showed a SI

lower than that of the control, which could indicate

pro-oxidant activity linked to the garlic product. The

antioxidant activity ofAlliumplants has been attributed

to a group of sulphur-containing compounds, from

which allicin (diallyl thiosulphinate) appeared to be

the main component (Kim et al., 1997). Animal tests

have shown that in low concentrations, allicin can be

responsible for the antioxidative properties of garlic,

although it can also act as a pro-oxidant in high concen-

trations (Lawson, 1998). Also, controversial results

about antioxidant properties of garlic extracts have been

found to be dependent on the sample extraction method.

These differences have been attributed to the spectrum

of compounds extracted with the different solvents used

(Nuutila et al., 2003; Yin & Cheng, 1998).The extracts with the highest (P< 0.05) antioxidant

activity were those obtained from rosemary (OR, WR,

OWR). There were no significant differences (P> 0.05)

between the oil extract of rosemary (OR) and the water

miscible rosemary extract (WR), which showed the high-

est SI. However, the oil and water miscible rosemary ex-

tract (OWR) had a lower SI.

Citrus extracts (LF, OF) showed antioxidant activity

but lower (P< 0.05) than rosemary extracts (OR, WR,

OWR). The SI of orange extract (OF) was higher

(P< 0.05) than lemon extract (LF).

The antioxidant activity of phenolic compounds from

plants is well known (Pokorny, 1991; Shahidi, 2000).

This activity has been mainly attributed to flavonoidsand ascorbic acid in citrus fruits (hesperidin, neohesperi-

din and eriocitrin) and to carnosol and rosmarinic acid

in rosemary (Schwarz et al., 2001). All of these polyphe-

nolic compounds have the ability to act as antioxidants

by a free radical scavenging mechanism and also

through their known ability to chelate transition metals

(inactivation of iron ions) (Martn et al., 2002).

3.2. Antibacterial activity of natural extracts

The antibacterial activity of natural extracts against

the spoilage bacterial strains tested is shown in Table 3.

The water (control) was ineffective. Lemon extracts

(LF), orange extracts (OF) and garlic extracts (GR)

were only active against one, two and three bacterial

strain/species, respectively. On the other hand, rosemary

extracts (OR, WR, OWR) were active against all bacte-

ria tested. From rosemary extracts studied, the oil misci-

ble extract (OR) showed the highest inhibitive effect

against each bacterium. Water miscible extract (WR)

showed higher antibacterial effect than water and oil

miscible extract (OWR) for all bacteria tested except

for Brochothrix spp. For all rosemary extracts (OR,

WR, OWR), Brochothrix spp. were the most sensitive

bacteria. Some reports found that the most apolar phe-nolic compounds from rosemary extracts are presuma-

bly responsible of their antibacterial activity (Del

Campo et al., 2000; Karamanoli, Vokou, Menkissoglu,

& Constantinidou, 2000).Davidson (1993)reported that

gram-positive bacteria are generally more susceptible to

nonpolar phenolic compounds than gram-negative

microorganisms. This could explain some of the

Table 3

Inhibition effects of natural extracts tested against spoilage bacteria

Spoilage bacterial strains Diameter of the zones of inhibition in mm (6 mm well)

OR WR OWR GR OF LF

Br. thermosphactaCRA 7883 28.1 19.5 23.8

Br. thermosphactaCRA 7884 25.5 19.5 23.8

Br. thermosphactaCRA 3235 26.4 24 25.1

L. innocua4202 20.5 17.4 14.2 8.5 7.0

L. monocytogenes5105 25 19.5 15.4 10.5

Lb. sake 550 21.4 13.4 11.19

Lc. mesenteroides subsp mesenteroides 824 21 16.6 13.8

Lc. mesenteroides subsp dextranicum 882 21 15.4 14.2

Lb. carnosum 558 26.2 23 14.3

Lb. curvatus 860 23.1 16.2 15.4 10.5

Lb. lactis FMRD 492 21.8 18.2 13 7.5 8.5

, no inhibition.

Table 2

Stability index of each natural extract determined by the Rancimat

method

Extract Stability indexx

Control (pork lard without extract) 1.00a

Duralox (WR) 1.82b

Herbalox Type HT-O (OR) 1.78b

Herbalox Type W (OWR) 1.46c

Lemon extract (LF) 1.19d

Orange extract (OF) 1.30e

Aquaresin garlic (GR) 0.69f

af Means within a column with different letters are significantly dif-

ferent (P< 0.05).x Stability index = induction time (conductivity curves) of the

sample/induction time of the control.



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differences in the antibacterial activity between the rose-

mary extracts studied (OR, WR, OWR), if we assume

that the oil extract (OR) is richer in nonpolar phenolic

compounds than the others.

Frazier (1980)reported that citrus bioflavonoids also

had antimicrobial properties. These compounds have

reportedly wide-ranging antimicrobial properties effec-tive against a broad range of human pathogens, fungi

and food spoilage organisms (Cho, Seo, Choi, & Joo,

1990; Harich, 1997; Morgan, Andersen, & Hankinson,

1971).

3.3. Antibacterial and antioxidant properties of natural

extracts in meatballs

The effect of the natural extracts on lipid oxidation of

cooked meatballs during storage is shown inFig. 1. The

analysis of variance for the TBARS data indicates that

the TBA values were significantly affected (P< 0.05)

by both the storage period and the extract treatments.

Initial (day 1) TBA values for all extract samples were

significantly lower than those for the control

(P< 0.05). These results suggest that these antioxidants

retarded lipid oxidation during and immediately after

cooking. These results agree with that reported by

Ahn, Grun, and Fernando (2002)and Fernandez-Lopez

et al. (2003) for other natural antioxidants applied to

cooked beef. Sato and Hegarty (1971) reported that

non-heme iron was the active catalyst in cooked meats.

Chen et al. (1984) also reported that iron was released

from heme pigments during cooking and proposed that

the resultant increase in non-heme iron was responsiblefor lipid oxidation.

At the end of storage time (day 12) all treatments re-

sulted in significantly lower (P< 0.05) TBA values when

compared to the control, which indicates that all the

tested natural extracts added to meatballs showed anti-

oxidant properties. The product samples with rosemary

extracts (OR, WR, OWR) showed the lowest (P< 0.05)

TBA values at each time of storage. Only treatments

with orange extracts (OF) and rosemary water miscible

extracts (WR) maintained (P> 0.05) the initial TBA val-

ues during the 12 day storage period, and no differences

were found between them (P< 0.05). Samples treated

with rosemary oil and water miscible extracts (OWR)had slightly increased TBA values only during the first

6 days of storage and became stable after that period.

The products with added lemon extracts (LF) reached

higher (P< 0.05) TBA values than those with orange ex-

tracts (OF) by the end of storage. Consistently, this dif-

ference in antioxidant properties between orange and

lemon extracts (OF and LF) has also been detected by

using the Rancimat method, as explained before.

All colour coordinates showed differences (P< 0.05)

between treatments and storage days, except yellowness

which only showed differences between treatments

(Table 4). In all samples,lightnessincreased with storage

time (P < 0.05), and the highest values of L* were ob-

tained in control samples. Some authors reported that

this increase could be related to the increase in MMb

formation (Anton et al., 1993; Genot et al., 1991). These

results suggests that the presence of antioxidant com-

pounds in the natural extracts could retard MMb for-

mation in meatballs and so L* values decreased. For

this reason, it could be expected that treatments with or-

ange extracts (OF) and rosemary water miscible extracts

(WR) would have lowest lightness values because they

had the highest antioxidant capacity (Table 2), but in

this study, treatments with lemon extracts (LF) and or-

ange extracts (OF) showed the lowest L* values. Thiscould be explained by the increased water retention

associated with hygroscopic materials, and because

these extracts were prepared as a dry powder, they have

absorbed free water within the product, subsequently

decreasing lightness values. This relation between free

water and lightness in meat and meat products has been

reported by several authors (Fernandez-Lopez, Perez-

Alvarez, & Aranda-Catala, 2000; Kauffman, Warner,

& Russell, 1991).

1

1,5

2

2,5

3

3,5

4

1 6 9

Time (days)

elpmas

gk/AMgm(ABT

12

)

Control OR WR OWR LF OF

Fig. 1. Rancidity (TBA) evolution in meatballs with different natural

extracts added, during storage time. For sample denomination see

Table 1.

Table 4

Effect of natural extracts on colour parameters of meatballs during

storage at 8 C

Treatment Lday 1 L

day12 a

day1 a

day12 b

day1 b

day12

Control 42.26ax 46.97ay 9.52ax 7.13ay 8.88ax 9.14ax

OR 42.32ax 44.73by 9.53ax 8.33by 8.78ax 9.07ax

WR 42.48ax 44.61by 9.39ax 8.42by 8.50ax 8.78ax

OWR 42.25ax 44.89by 9.33ax 8.45by 9.11ax 9.22ax

LF 39.46bx 42.08cy 10.22bx 8.83by 10.30bx 10.34ax

OF 39.28bx 41.57cy 10.37bx 8.92by 10.29bx 10.33ax

For sample denomination seeTable 1.a,b Means within a column with different letters are significantly dif-

ferent (P< 0.05).x,y Means within a row with different letters are significantly different

(P< 0.05).



7/10

In all samples redness decreased as the storage time

progressed (P< 0.05) but red colour (a* values) of the

control sample faded very rapidly. Phillips, Mancini,

Sun, Lynch, and Faustman (2001) reported a decrease

in a* values of cooked ground beef linked to storage

time. This is not surprising as meat which has been

stored longer would be expected to have predominantlyeither OMb or MMb, as opposed to deoxymyoglobin

(DMb), which in turn would predispose the meat to a

faster browning rate (Hunt, Sorheim, & Slinde, 1995,

1999). At the end of storage (day 12) a* values of the

control samples were lower (P< 0.05) than day 1, and

lower that of antioxidant treatments. Several authors

have studied the effect of different antioxidants on the

colour of meat and meat products (Higgins, Kerry,

Buckley, & Morrisey, 1998; Lee, Hendricks, & Corn-

forth, 1998) and have reported that meat oxidation de-

creases a* values. Therefore, it was expected that

samples with citrus extracts (LF and OF), which were

the treatments with lower antioxidant activity (Table 2),

would have the lowesta* values. Probably, this decrease

in redness associated with the oxidation process is being

compensated by the presence of red component pig-

ments such as carotene in these citrus extracts. To con-

firm this, a significant effect of carotenes increasing a*

values by about one unit was observed after preparation

of the samples (day 1) (Table 4). If adjustments were

made by subtracting the contribution of added pig-

ments, the results would be consistent with previous

observations.

In all samples yellowness values were not modified

(P< 0.05) by storage time. Therefore, the differences inb* values observed between treatments incorporating

citrus extracts (LF, OF) and the others, can be attrib-

uted to the presence of pigments in the citrus extracts

and not to the oxidation processes.

In this work, the presence of coliforms, moulds and

yeasts, and psychrotrophic microorganisms was not de-

tected in any cooked meatball samples, regardless of

storage time. This product was prepared following safe

practices resembling commercial aseptic conditions,

with a heat treatment that guaranteed pasteurization

effectively inactivating vegetative cells and then stored

under conditions that would limit the growth bacterial

groups. Cross contamination from the environment

(i.e. airborne or food handlers) or the survival of spores

or resistant cells was possible, as it is in commercial

operations. Lactic acid bacteria were detected at low lev-

els in the control and samples with rosemary water and

oil miscible extracts (OWR) from day 1 (data not

shown). Some spore formers and heat resistant strains

which have been linked with spoilage of meats (includ-

ing VP and MAP) are likely to contribute to LAB

counts (Borch, Kant-Muermans, & Blixt, 1996; Kuri,

1998). After 12 days storage, the growth of LAB to lev-

els of 103 log cfu/g was similar (P> 0.05) for control

samples and treatments with rosemary extracts (OR,

WR and OWR) (Table 5). These bacterial groups were

not detected in samples from any treatment with citrus

extracts (LF and OF) during storage time. Despite the

presence of lactic acid bacteria, there was no evidence

of strong lactic fermentation in any product, as con-

firmed by very low (


8/10

From the eight sensory attributes evaluated, only

three (aroma rancidity, surface lime and overall accept-

ance) provided useful information on the changes due to

treatment and storage day, while for brightness, citrus

aroma, putrefaction and sour aroma no differences

(P> 0.05) were detected for any of the factors. The

products were fairly stable for a week, with some differ-ences becoming apparent between day 6 and day 9. In

Fig. 2it can be observed that at the end of the storage

period (day 12), the differences in rancidity perception

between treatments (P< 0.05) are consistent with differ-

ences measured as TBA values (Fig. 1). One exception is

the lower score for orange extracts (OF) in comparison

to lemon extracts (LF). This correlation between the

sensory and the chemical methods is less evident at

day 6, because at this point, changes in TBA values seem

to show differences before panellist could perceive the

increased rancidity. While these results suggest that the

panellist may not be sensitive enough to detect differ-

ences of TBA values below a threshold, sensory scores

provided better discrimination at day 9 in comparison

to TBA values. Interestingly, samples with citrus ex-

tracts (LF, OF) were scored at equivalent terminal levels

from day 9. Another factor to consider is the presence of

off flavours in lemon extracts (LF), which could contrib-

ute to perceived rancidity, but are not identifiable by the

TBA reaction. Overall acceptability scores for each

treatment (Fig. 2) showed an inverse relation with

TBA values and rancidity perception (Figs. 1 and 2,

respectively). Product surface slime was not detected in

any treatment during the first days of storage, but incontrol samples it was detected at day 9, increasing by

day 12, when it was also detected in samples from treat-

ments with rosemary extracts (OR, WR and OWR). No

perception of surface slime was detected in citrus treat-

ments (OF, LF) during storage time (results not shown).

These observations agree with LAB enumeration results

(Table 5).

The only attribute that showed differences (P< 0.05)

between treatments but not between storage days was

aroma off odour, which suggests that it was mainly

due to the type of extract added and not to the deterio-

ration of meatballs during storage. So, this off odour

perception can be attributed to the peculiar composition

of each extract, which is detected at its incorporation

and was not modified during storage. The highest

(P< 0.05) scores for off odours were obtained for citrus

treatments (LF, OF), the lowest for control samples and

there were not differences (P> 0.05) in off odours per-

ception between rosemary treatments (OR, WR,

OWR). Therefore, it can be observed that this off odour

perception may not to have any influence upon the over-

all acceptability of the products.

4. Conclusions

The use of rosemary extracts has proved to be

effective as an antioxidant in cooked swedish-style

meatballs. The application of rosemary and citrus ex-

tracts improved the acceptability of the product. The

application of rosemary preparations could be useful

to control the development of rancidity and off fla-

vours, while orange and lemon serve the same pur-

pose, but may have some additional effects such as

water binding that need to be managed. Some anti-

bacterial activity was detected, but additional meas-

ures would be needed to control spoilage. The effect

of antibacterial interaction with fat and the physical

availability and compatibility of the fatty and lean

phases and the active compounds need to be estab-

lished in order to control better the deterioration of

meat products.

Acknowledgement

Funding for work at the University of Plymouth was

enabled by a grant by the Seale Hayne Educational

Trust. JFL visit to U.P. was funded by the Generalitat

1

2

3

4

5

6

7

1 6 9

Time (days)

Rancidity(aroma)

12

Control OR W R OW R LF OF

1

2

3

4

5

6

7

1 6 9

Time (days)

Overallacceptability

12

Control OR W R OW R LF OF

(a)

(b)

Fig. 2. Evolution of sensory scores for perception of rancidity aroma

(a) and overall acceptability (b) of meatballs during storage time. For

sample denomination seeTable 1.



9/10

Valenciana and LACS was within a Socrates exchange

programme. Linda Thomas (Aplin & Barrett Ltd., Da-

nisco-Cultor, Beaminster, UK) kindly provided some

of the bacterial strains.

References

Afifi, A. A., & Azen, S. P. (1979). Statistical analysis. A computer

oriented approach. London: Academic.

Ahn, J., Grun, I. U., & Fernando, L. N. (2002). Antioxidant properties

of natural plant extracts containing polyphenolic compounds in

cooked ground beef.Journal of Food Science, 67, 13641369.

Anton, M., Salgues, C., & Renerre, M. (1993). Etude des relations

oxidatives entre les lipides membranaires et la myoglobine in vitro.

Science Alimentarie, 13, 261274.

Borch, E., Kant-Muermans, M. L., & Blixt, Y. (1996). Bacterial

spoilage of meat and cured meat products.International Journal of

Food Microbiology, 33, 103120.

Borch, E., & Molin, G. (1988). Numerical taxonomy of psychrotrophic

lactic acid bacteria from prepacked meat and meat products.

Antonie van Leeuwenhoek, 54, 301323.

British Standards BS 5929: Part 4. (1986a). British standard methods

for sensory analysis of foods. Part 4. Flavour profile methods.

British Standards Institution, London.

British Standards BS 5929: Part 1. (1986b). British standard methods

for sensory analysis of foods. Part 1. General guide to methodol-

ogy. British Standards Institution, London.

Chastain, M. F., Hufman, D. L., Hsieh, W. H., & Cordray, J. C.

(1982). Antioxidants in restructured beef/pork steaks. Journal of

Food Science, 47, 17791782.

Chen, H. M., Muramoto, K., Yamauchi, F., & Huang, C. L. (1996).

Natural antioxidants from rosemary and sage. Journal of Food

Science, 42, 11021104.

Chen, C. C., Pearson, A. M., Gray, J. I., Fooladi, M. H, & Ku, P.

(1984). Some factors influencing the nonheme iron content of meat

and its implications in oxidation. Journal of Food Science, 49,581584.

Cho, S. H., Seo, I. W., Choi, J. D., & Joo, I. S. (1990). Antimicrobial

and antioxidant activity of grapefruit seed extract on fishery

products. Bull Korean Fish Society, 23, 289296.

Cuvelier, M. E., Richard, H., & Berset, C. (1996). Antioxidative

activity and phenolic composition of pilot-plant and commercial

extracts of sage and rosemary. Journal of the American Oil

ChemistsSociety, 73, 645652.

Daeschel, M. A. (1992). Procedures to detect antimicrobial activities of

microorganisms. In B. Ray & M. A. Daeschel (Eds.), Food

preservatives of microbial origin (pp. 155176). Boca Raton, FL:

CRC Press.

Davidson, P. M. (1993). Parabens and phenolic compounds. In P. M.

Davidson & A. L. Branen (Eds.), Antimicrobials in foods

(pp. 263306). New York: Marcel Dekker.Davies, E. A., Milne, C. F., Bevis, H. E., Potter, R. W., Harris, J. M.,

Williams, G. C., et al. (1999). Effective use of nisin to control lactic

acid bacterial spoilage in vacuum-packed bologna-type sausages.

Journal of Food Protection, 62(9), 10041010.

Dean, S. G., & Svoboda, K. P. (1989). Antimicrobial activity of

summer savory (Satureja hortensis L.) essential oil and its constit-

uents. Journal of Horticultural Science, 64, 205210.

Decker, E. A., Chan, W. K. M., Livisay, S. A., Butterfield, D. A., &

Faustman, C. (1995). Interactions between carnosine and the

different redox states of myoglobin. Journal of Food Science, 60,

12011204.

Del Campo, J., Amiot, M. J., & Nguyen-The, C. (2000). Antimicrobial

effect of rosemary extracts. Journal of Food Protection, 63,

13591368.

Doyle, M. E. (1999). Use of other preservatives to control Listeria in

meat. American Meat Institute.

Elangovan, V., Sekar, N., & Govindasamy, S. (1994). Chemoprotec-

tive potential of dietary bipoflavonoids against 20-methylcholanth-

rene-induced tumorigenesis.Cancer Letters, 87, 107113.

Farag, R. S., Daw, Z. Y., Hewedi, F. M., & El-Baroty, G. S. A. (1989).

Antimicrobial activity of some Egyptian spice essential oils.Journal

of Food Protection, 52, 665667.

Fernandez-Gines, J. M., Fernandez-Lopez, J., Sayas-Barbera, E.,

Sendra, E., & Perez-Alvarez, J. A. (2003). Effects of storage

conditions on quality characteristics of bologna sausages made

with citrus fiber. Journal of Food Science, 68, 710715.

Fernandez-Lopez, J., Fernandez-Gines, J. M., Aleson-Carbonell, L.,

Sayas-Barbera, E., Sendra, E., & Perez-Alvarez, J. A. (2004).

Functional compounds from citrus by-products and their applica-

tion in meat products. Trends in Food Science and Technology, 15,

176185.

Fernandez-Lopez, J., Perez-Alvarez, J. A., & Aranda-Catala, V.

(2000). Effect of mincing degree on colour properties in pork meat.

Color Research & Application, 25, 376380.

Fernandez-Lopez, J., Sevilla, L., Sayas-Barbera, M. E., Navarro, C.,

Marn, F., & Perez-Alvarez, J. A. (2003). Evaluation of the

antioxidant potential of hyssop (Hyssopus officinalis L.) and

rosemary (Rosmarinus officinalisL.) extract in cooked pork meat.

Journal of Food Science, 68, 660664.

Frazier, S. F. (1980). Antimicrobial composition of matter from

naturally occurring flavonoid glycosides, US Pat. 4,238,483.

Genot, C., Borel, M. N., Metro, B., Gandemer, G., & Renerre, M.

(1991). Enhancement of myoglobin autoxidation induced by

phospholipids extracted from beef muscles of various metabolic

types. In Proceedings of the 37th International Congress of Meat

Science and Technology (pp. 356359), 16 September, Kulmbach,

Germany.

Gerhardt, P., Murray, R. G. E., Costilow, R. N., Nester, E. W., Wood,

W. A., & Phillips, G. B. (1994). Manual of methods for general

bacteriology. Washington: American Society for Microbiology.

Harich, J. (1997). Antimicrobial grapefruits extracts. US Pat.

5,631,001.Harris, J. C., Cottrell, S. L., & Plummer, S. (2001). Antimicrobial

properties of Allium sativum (garlic). Applied Microbiological

Biotechnology, 57, 282286.

Higgins, F. M., Kerry, J. P., Buckley, D. J., & Morrisey, P. A. (1998).

Effect of dietary a-tocopheryl acetate supplementation on

a-tocopherol distribution in raw turkey muscles and its effect on

the storage stability of cooked turkey meat. Meat Science, 37,

373383.

Houlihan, C. M., Ho, C. T., & Chang, S. S. (1984). Elucidation of the

chemical structure of a novel antioxidant, rosmaridiphenol,

isolated from rosemary. Journal of the American Oil Chemists

Society, 61, 10361039.

Houlihan, C. M., Ho, C. T., & Chang, S. S. (1985). The structure of

rosmariquinone- A new antioxidant isolated from Rosmarinus

officinalis L. Journal of the American Oil Chemists

Society, 62,9698.

Hunt, M. C., Acton, J. C., Benedict, R. C., Calkins, C. R., Cornforth,

D. P., Jeremiah, L. E., et al. (1991). Guidelines for meat color

evaluation. Chicago: American Meat Science Association and

National Live Stock and Meat Board.

Hunt, M. C., Sorheim, O., & Slinde, E. (1995). Effects of myoglobin

form on internal cooked color development in ground beef. In

Proceedings of the 41st International Congress of Meat Science

Technology (pp. 394395), 2025 August, San Antonio, USA.

Hunt, M. C., Sorheim, O., & Slinde, E. (1999). Color and heat

denaturation of myoglobin forms in ground beef. Journal of Food

Science, 64, 847851.

IFST. (1993). Shelf life of foods Guidelines for its determination and

prediction. London: Institute of Food Science and Technology.



10/10

Karamanoli, K., Vokou, D., Menkissoglu, U., & Constantinidou, H. I.

(2000). Bacterial colonization of phyllosphere of mediterranean

aromatic plants.Journal of Chemical Ecology, 26, 20352048.

Kauffman, R. G., Warner, R. D., & Russell, R. L. (1991). Variation in

lightness and exudation among ten porcine muscles. InProceedings

of the 37th International Congress of Meat Science Technology(pp.

135138), Kulmbach, Germany.

Kim, S. M., Kubota, K., & Kobayashi, A. (1997). Antioxidative

activity of sulphur-containing flavour compounds in garlic. Bio-

science Biotechnology Biochemistry, 61, 14821485.

Kuri, V. (1998). Lactic Acid Bacteria and Salmonellae from Mexican

pork products: Characterization and antagonism. Ph.D. Thesis.

The Queens University of Belfast.

Kuri, V., Collins, M. A., & Madden, R. H. (1998). Stability and

antibacterial activity of bacteriocin preparations in pork model. In

Proceedings of the 44th International Congress of Meat Science and

Technology (pp. 342343), 30 August to 4 September 1998,

Barcelona, Spain.

Kuri, V., Ponce, E., & Guerrero, I. (1994). Physicochemical charac-

teristics of fermented sausage extended with carbohydrate materi-

als. Journal of Muscle Foods, 5, 119135.

Lampe, J. W. (1999). Health effects of vegetables and fruit: assessing

mechanisms of action in human experimental studies. American

Journal of Clinical Nutrition, 70, 475S490.

Lario, Y., Sendra, E., Garca, J., Sayas-Barbera, E., Fernandez-Lopez,

J., & Perez-Alvarez, J. A. (2003). Preparation of high dietary fiber

powder from lemon juice by-products. InNew Functional Ingredi-

ents and Foods Abstract Book (P1-G20), 911 April 2003, Copen-

hagen, Denmark.

Laubli, M. W., & Bruttel, P. A. (1986). Determination of the oxidative

stability of fats and oils: comparison between the Active Oxygen

Method (AOCS Cd 1257) and the Rancimat Method. Journal of

the American Oil Chemists Society, 63, 792795.

Lawson, L. D. (1998). Garlic: a review of its medicinal effects and

indicated active compounds. In L.D. Lawson, R. Bauer (Eds.),

Phytomedicines of Europe: chemistry and biological activity (ACS

Symposium series No. 691 (pp. 176209). Washington, DC:

American Chemical Society.Lee, B. J., Hendricks, D. G., & Cornforth, D. P. (1998). Antioxidant

effect of carnosine and phytic acid in a model beef system.Journal

of Food Science, 63, 394398.

Loliger, J. (1991). The use of antioxidants in foods. In I. O. Aruoma &

B. Halliwell (Eds.), Free radical and food additives (pp. 121131).

London: Taylor & Francis.

Martn, F. R., Frutos, M. J., Perez-Alvarez, J. A., Martnez-Sanchez,

F., & Del Ro, J. A. (2002). Flavonoids as nutraceuticals: structural

related antioxidant properties and their role on ascorbic acid

preservation. In Atta-Ur-Rahman (Ed.), Studies in natural

products chemistry (Vol. 26, pp. 324389). Amsterdam: Elsevier

Science.

Morgan, D. R., Andersen, D. L., & Hankinson, C. L. (1971). Process

of inhibiting staling of milk prior to sterilization, US Pat.

3,615,717.

Mouly, P. P., Arzouyan, C. R., Gaydou, E. M., & Estienne, J. M.

(1994). Differentiation of citrus juices by factorial discriminant

analysis using liquid chromatography of flavonone glycosides.

Journal of Agricultural and Food Chemistry, 42, 7079.

Nakatani, N., & Inatani, R. (1984). Two antioxidant diterpenes from

rosemary (Rosmarinus officinalisL.) and a new revised structure for

rosmanol. Agricultural Biological Chemistry, 48, 20812085.

NCCLS. Global Consensus Standardization for Health Technologies

(1990). Quality assurance for commercially prepared microbiological

culture media. Approved standard document M22-A, Vol. 10, No.

14.

Nuutila, A. M., Puupponen-Pimia, R., Aarni, M., & Oksman-

Caldentey, K. M. (2003). Comparison of antioxidant activities of

onion and garlic extracts by inhibition of lipid peroxidation and

radical scavenging activity.Food Chemistry, 81, 485493.

Pandit, V. A., & Shelef, L. A. (1994). Sensitivity of Listeria

monocytogenes to rosemary (Rosmarinus officinalis L). Food

Microbiology, 11, 5763.

Phillips, A. L., Mancini, R., Sun, Q., Lynch, M. P., & Faustman, C.

(2001). Effect of erythorvic acid on cooked colour in ground beef.

Meat Science, 57, 3134.

Pokorny, J. (1991). Natural antioxidant for food use.Trends in Food

Science and Technology, September, 223226.

Renerre, M., Anton, M., & Gatellier, P. (1992). Autoxidation of

purified myoglobin from two bovine muscles.Meat Science, 32,

331342.

Sato, K., & Hegarty, G. R. (1971). Warmed over flavor in cooked

meats. Journal of Food Science, 36, 10981102.

Schieber, A., Stintzing, F. C., & Carle, R. (2001). By-products of plant

food processing as a source of functional compounds recent

developments.Trends in Food Science and Technology, 12, 401413.

Schwarz, K., Bertelsen, G., Nissen, L. R., Gardner, P. T., Heinonen,

M. I., Hopia, A., et al. (2001). Investigation of plant extracts for

the protection of processed foods against lipid oxidation. Com-

parison of antioxidant assays based on radical scavenging, lipid

oxidation and analysis of the principal antioxidant compounds.

European Food Research Technology, 212, 319328.

Shahidi, F. (2000). Natural phenolic antioxidants and their foodapplications. Lipid Technology, 12, 8084.

Tarladgis, B. G., Watts, B. M., & Younathan, M. T. (1960). A

distillation method for the quantitative determination of malonal-

dehyde in rancid foods. Journal of the American Oil Chemists

Society, 37, 4448.

Tsao, S. M., & Yin, M. C. (2001). In-vitro antimicrobial activity of 4

dialyl sulphides occurring naturally in garlic and Chinese leek oils.

Journal of Medical Microbiology, 50, 646649.

Yin, M. C., & Cheng, W. (1998). Antioxidant activity of several Allium

members. Journal of Agricultural and Food Chemistry, 46,

40974101.

Yin, M. C., & Faustman, C. (1993). Influence of temperature, pH and

phospholipid composition upon the stability of myoglobin and

phospholipid: a liposome model. Journal of Agricultural and Food

Chemistry, 41, 853857.


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