RESEARCH OF THE PHYSICAL AND CHEMICAL PROPERTIES AND METHODS OF RED RICE (FOOD COLOUR) DETERMINATION IN SAUSAGE PRODUCTS Текст научной статьи по специальности «Химические технологии»

DOI: https://doi.org/10.21323/2414-438X-2020-5-4-23-28

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Available online at https://www.meatjournal.ru/jour Original scientific paper

RESEARCH OF THE PHYSICAL AND CHEMICAL PROPERTIES AND METHODS OF RED RICE (FOOD COLOUR) DETERMINATION IN SAUSAGE PRODUCTS

Natalia V. Rudometova, Irina S. Kim

All-Russia Research Institute for Food Additives — Branch of V. M. Gorbato Federal Research Center

for Food Systems of RAS, St. Petersburg, Russia

Keywords: food colours, monascus, red rice, sausage products, extraction, thin layer chromatography, high performance liquid chromatography, identification, spectrophotometric analysis

Abstract

In Russia, in the production of meat and sausage products, the food colour, named as Red rice, is use. Red rice is obtain by the cultivation the strains of the Monascus fungus on various carbohydrate substrates, for example rice. That Red rice may contain the mycotoxin citrinin, but neither the purity of the food colour nor the safety profile are regulated. The aim of this work was to study the physical and chemical properties of Red rice and to develop method for its determination in sausage products. The experiments were carried out on model and commercial samples of sausages. The samples were analyzed using spectrophotometry and high-efficiency planar and liquid chromatography. Spectrophotometric analysis revealed differences in solubility, coloring power, spectral characteristics, composition and ratio of pigments in commercial samples of Red rice. The extraction parameters of colouring substances from sausages model samples were determined. It has been established that Red rice is extracted with chloroform, acetone, ethanol and its aqueous solutions. Petroleum ether was proposed for degreasing sausages. Red rice from sausages was extracted with acetone because it did not extract synthetic colours and carminic acid. It was found, that 90% of Red rice is extracted from samples of sausages by double extraction with ultrasonic treatment with a power of 128 W. High performance thin layer chromatography method and high performance liquid chromatography method for the Red rice identification was proposed. The content of Red rice in the extracts was determined by spectrophotometric method. The developed method for the determination of Red rice was tested on commercial samples of sausage products.

Introduction

The current situation on the Russian market is characterizing by a significant amount of falsified food products and various food additives, including colours [1]. Most food colours are xenobiotics, therefore, it is necessary to control the use of colors in food production for safety reasons. In Russia red food colours Red rice, Carmines E120 and Ponceau 4R E124 are used in production of some meat and sausage products [2].

Red rice is obtained by fermenting various carbohydrate substrates, most often rice, by the strains of the fungus Monascus, which produce yellow and red substances: C21H2605 (Monascine), C23H30O5 (Ankaflavine), C21H2205 (Rubropunctatine), C23H2605 (Monascorubine), C21H23O4N (Rubropunctamine) and C23H27O4N (Monascorubramine), whose structural formulas are shown in Figure 1 [3,4].

Various genus of the Monascus are used to obtain the colour: Monascuspurpureus, Monascuspilosus, Monascus anka and Monascus ruber. The strain-producer and the conditions of its cultivation affect the content and composition of colour substances. But technical information about Monascus colours production is a commercial secret and therefore rarely published [5]. The colours are highly soluble in ethanol and slightly soluble in water. The color of its solutions depends on pH: orange at pH 3^4, red at pH 5^6, purple at pH 7^9. Monascus colour-

ing substances are fairly stable in 70% aqueous solution of ethanol. It is resistant to light, high temperatures and oxidation. Red rice is use in China as a food additive for over 2000 years [6].

It was found that Red rice contains the mycotoxin citrinin [7], but neither the criteria for its purity, nor the safety indicators in TR CU029/2012 are regulated, as well as the method for the determination of this food colours [2]. China is the main manufacturer and supplier of Red rice. However, the current Chinese National Standard does not set a maximum limit for citrinin in pigments produced by Monascus. Extensive testing of Red rice has not been conducted in the USA, but consumers are warned to avoid dietary supplements based on it due to possible myopathy and renal failure [5].

In the European Union the maximum level of citrinine (2 mg / kg) in Red rice preparations is regulate. This level ensures the possible exposure of citrinine from these drugs to well below the level of its nephrotoxicity (0.2 ^g / kg body weight) [8]. However, the results of Red rice various samples study showed that the content of citrinin can reach 7 ^g / kg of the product [9]. Acceptable daily intake of Red rice for humans has not been established. As the uncertainty about the carcinogenicity and genotoxicity of Red rice remains, it was decided to revise its maximum allowable level in foods [8, 10, 11].

FOR CITATION: Rudometova N. V., Kim I. S. Research of the physical and chemical properties and methods of Red rice (food colour) determination in sau-

sages products. Theory and practice of meat processing. 2020; 5(4): 23-28. https://doi.org/10.21323/2414-438X2020-5-4-23-28

Rubropunctatine

Monascorubine

.CtH-i

Monascine

Ankaflavine

Hac

Rubropunctamine

Monascorubramine

Figure 1. The structural formulas of Red rise colouring substances

The inadequate level of researching Red rice, as well as the possibility of falsification, which consists in replacing one colour with another, dictates the need to develop determination methods of colours in food products. The aim of this work was to study the physical and chemical properties of Red rice and to develop method for its determination in sausage producas.

Materials and methods

The objects oc the study were commercial samples of food colours Red Rice, Carmines E120, Ponceau 4R E124 and cooked smoked sausages: «Cervelat Muskatny», «Cer-velat Konyachny», «Cervelat Gubernsky», which were produced in accordance with the manufacturers' specifications.

Table 1. Conditions for colours assay by HPTLC method

The content of colours in the samples and extracts was determined by measuring the optical density of solutions at the maximums of light absorption at characteristic wavelengths by the spectrophotometry (SPh) method. The measurement was carried out on a SHIMADZU UV-1800 double-beam scanning spectro-photometee in the wavelength range of 300-700 nm against ths solvent. Mass fraction of colours in the object of study (in %) was calculated according to the forrmdas in the refeeence source [12]. The colours were identified by high performance thin layer chromatography (HPTLC) method on «Sorbfil» PET sheet plates (Table 1) and high performance liquid chromatography (HPLC) method.

№ Eluent composition, v/v Values Rf for colours

Ponceau 4R E124 Carmines E120 Red rice

0.43 ± 0.03

1 pyridine: 3-methyl-1-butanol: 2-methylpropanol-1: ethanol: 0.33 ± 0.03 0.00 0.53 ± 0.03

ammonia 25% (3: 3: 3: 4: 4) 0.64 ± 0.03 0.79 ± 0.03

2 acetone: ethanol: ammonia 25%: water (7: 3: 0.05: 3) 0.89 ± 0.03 0,00 0.49 ■ 0.64 0.76 ± 0.03 0.84 ± 0.03

0.74 ± 0.03 0.93 ± 0.03

Also the colours were identified by reverse phase HPLC on a chromatograph VARIAN920 — LC with a diode array detector: column Polaris C8A 150x4.6 mm (5^m), temperature 28 °C, flow rate 0.6 cm3/min. Eluent is a mixture of 0.02 M solution of sodium acetate and acetonitrile in a volume ratio of 90:10. Sample volume is (10 - 50) mm3, detector waveleng(h at 500 nm, time of anaiysis is 10 minutes. The samples were filtered through a filter with a pore diameter of 0.45 |im. The obtained chromatograms were processed using the GALAXIE program.

Sample preparation for the determination of colours in cooked smoked sausages was cirried out in accordance with GOST R ISO 13496-2013. Sijmples were defatted with chloroform or petroleum ether and centrifuged. The colours were extracted from defatted cooked smoked sausages with constant stirring ait a temperature from 20 °C to 60 °C and varying the extraction time from 10 to 60 min. Extraction was performed in a stirring device with a rotation speed 120 rpm, in a Bandelin Sonorex ultra-tonic bath at a power of 128 W and 160 W, and on a magnetic stirrer 0,70 -I

0,60 -

0,50 -

$ 0,40 --o 0,30 -

"s

J 0,20 J 0,10 -

wiih a stirrer rotation speed of400 to 1200 rpm. For extraction we used: chloroform, petroleum ether, cyclohexane, hexane, acetone, ethyl alcohol, its aqueous solutions with a mass fraction of ethyl alcohol 50% and 70%), as well as mixtures of these solvents with the ratio sample: extractant 1:4. The resulting extracts were centrifuged for 15 minutes et 6000 rpm. In the supernatant the colours content was determined by the spectrophotometric method.

All experimental measurements were performed three times. Analfais of variance oC tire; obtained data was carried out by Microsoft Excel with a significant difference at P 0.05. Graphical dependencies were obtained using Mi-eroroft Excel software.

Results and discussion

SPh analysis of commercial samples of Red rice, carried out in accordance with the method [12], shows that the samples differ in their coloricg power, spectral characteristics (Figure 2), composition and quantitative ratio of pigments (Figure 3). The pigment content varied from

0,00

350

450

550

650

—•—70 % Et

Sample 1

Figure 2. Absorption spectra of different Red rice commercial samples 220 i

170 -

120 -

70 -

20 -

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_

"3° > > > ^ ^ & ^VV ^ <</

Yellow Orange ■ Red

Sample 1

Figure 3. Colour co mposition of differentRed rice comm ercial samples

Wavelength, nm Aceton —•— Chloroform

350 -70 % Et

450

550 650 Wavelength,nm

•— Aceton

Sample 2

■ Chloroform

o

180 -| 160 -140 -120 -100 -80 -60 -40 -20 -0

////////

S

I

_

^

Yellow ■ Orange

Sample 2

I Red

(60 ± 2)% to (90 ± 2)%. Experiments show that chloroform and acetone extract all of the Red rice coloring substances. Petroleum ether extracts only a small amount of yellow and orange pigments, which will not affect the subsequent identification of the Red rice. Therefore, the petroleum ether is the best for degreasing of the sau0ages. From the reference source it is known that the isolation of Red rice from various matrices is carried out with water-ethanol solutions at the temperature range from 330 °C to 60 °C [8, 11, 13]. However, a study of the solubility of colours show, that Carminic acid and Ponceau 4R, as well as Red rice, dissolve in water-ethanol solutions, and these colours have very similar spectral characteristics (Figures 4, 5).

Carminic acid and Ponceau 4R is not extracted in acetone, therefore, it was proposed to isolate Red rice from defatted sausages with acetone.

The extraction process of colours was studied on model samples. Analysis of the sample «Cervelat» Mus catny» showed that it does not contain any colours, thecefore, model samples were made on its basis. Colours, celite or sodium sulfate, or quartz sand were added to the model samples. Then the sample was triturated until it became homogeneous. It was shown that celite and sodium sulfate was adsorbed colours irreversibly. Therefore, quartz saed ■was chosea for grinding the samples. Its known, vhat ultrasonic action is increasing the extraction of different substances from plant and animal matrices [14, 15]. The results

1,00 -

0,80 -

0,60 -

ne 1

-a 0,t0 1

u

a O 0,20 -

0,00 -

350

t5t

550 650

Wavele ngth, nm

—•— Red rice —9— Carminic acid —9— Ponceau tR Figure 4. Absorption spectra of colours in 50% water-ethanol solution

(Figure 6) showed that 90% of the Red rice was extracted from the samples in two stages of extraction with ultrasonic treatment.

Aooording to the reference sources, the quantitative determination of Red Rice, as a rule, is carried out by SPh and HPLC methods [3,4,5,7,8,9,10,16,17,18]. Based on the results of the identification of colours by HPTLC, two elu-tion systems were selected (Table 1). These eluents correctly identify Red rice in the presence of synthetic colours and Carmine E120 (Figure 7).

According to the experimental data obtained, proposed HPLC method also allows correct identification of colours in sausage products (Figure 8). The retention time of the colours was (4.5 ± 0.2) min for Carminic acid, for Ponceau 4R — (7.3 ± 0.2) min,for Red rice — (3.3 ± 0.2) min. Thus, the methods of identification oe Red rice, Carmines and Ponceau 4R with their possible simultaneous presence in the anclyze d s ample have been determine d.

The developed method was tested on samples of boiled smoked sausages "Cervelat" Konyachny" and "Cervelat "Gube rnsky". F ood colours Carmine E120 in the amount of (4 .0 ± 0.4) mg/kg and Red rice in the amount of (32 ± 3) mg/ kg were found in the sample "Cervelat" Konyachny ". No colours were found in the sample "Cervelat" Gubervski. ". S ampl e analyois rasults are consistent with) the information on the sample label.

80 60

2 40

cS

15 20

W

0

1 2 3

■ 1 stage

1 — without ultrasonic treatment,

2 — ulteastnic treatment wiah a pttwer of 1288 W,

3 — ultrasonic treatment with a power of 160 W

Figure 6i Influenca of ultrasonic treatment on the acetone extraction of Red rice from model samples

1,00 0,90 0,80 0,70 -0,60 -0,50 0 0,40 0 0,30

0,20 0 0,10 0,00

Wavelengt6, nm —•— Red rite —1•— Carminic atiS Wa— Ponceau 4R Figure 5. Absorption spectra of colours in 70% water-ethanol solution

3

u £

s

ca

a

O

1 2 3 Eluent 1

5 6 7 8 9 Eluent 2

Figure 7. The identification results of Carminic tiid (1), Ponce au 4R (2), Red rice (3,4,7,8,9) and a mixture of Carminic acid and Ponceau 4R (5,6) obtained by HPTnC method

4

A

B

C

D

nriAÜ

. ,__(,

______ [ïfT RT [min]

0 1 2 3 4 5 6 7 8 9 1(1 11 (2 13 14 15

Figure 8. The identification results of Ponceau 4R (A), Carminic acid (B) by HPLC method

Conclusion

The process of extracting food colors has been studied on model samples of cooked smoked sausages. The extraction parameters of Red ri cefrom s ausages are determined. Spectrophotometric rnalysis revealed differences in solubility, coloring power, spectral characteristics, compoiiti on and ratio of pigments in commercial

TTlflfr

1 2 3 4 S 6 7 8 9 10 11 12 13 14 1E

Red rice (C) and a mixture of Carminic acid and Red rice (D), obtained

samples of Red rice. It is propose d to use petroleum ether for defatting sausages, and acetone for the extraction of Red rice. Methods of spectrometric and chro-matographic analysis have been developed to determine the Red rice in cooked smokeO saueages. The developed method was tested on commercial samples of cooked smoked sausagrs.

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AUTOOR INFORMATION

Natalia V. Rudometova — candidate of chemical sciences, leading research scientist, laboratory of physical and chemical research methods, All-Russia Scientific Research Institution for Food Additives — Branch of V. M. Gorbatov Federal Research Center for Food Systems of RAS, 191014, Sankt-Petersburg, Liteyniy Prospect, 55. Tel. +7-812-272-75-78. E-mail: [email protected] ORCID: https://orcid.org/0000-0002-9414-6806 * corresponding author

Irina S. Kim — junior research scientist, laboratory of physical and chemical research methods, All-Russia Scientific Research Institution for Food Additives — Branch of V. M. Gorbatov Federal Research Center for Food Systems of RAS, 191014, Sankt-Petersburg, Liteyniy Prospect, 55. Tel. +7-812-272-56-26. E-mail: [email protected]

All authors bear responsibility for the work and presented data.

All authors made an equal contribution to the work.

The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism. The authors declare no conflict of interest.

Received 09.09.2020 Accepted in revised 06.12.2020 Accepted for publication 20.12.2020