The effect of ascorbic acid, storage period and packaging material on the formation of volatile N-nitrosamine in sausages

Abstract

This study aims to determine the amounts of seven volatile N-nitrosamine (VNA) derivatives which are in the risk group, in processed sausages. It also aimed to investigate the effects of the amount of added ascorbic acid, on the VNA level during the sausage manufacturing processes. For this purpose, meat doughs were prepared with two different levels of ascorbic acid. These meat doughs were put into the production process and packaged in two different packages. Thus, VNA derivatives and their amounts were determined according to production stages (heat treatments), packaging method (vacuum, MAP), storage process (Day 1, 7, 15, 30, 60, 90). As a result, it was found that the sausage product carries risk of VNA formation from the beginning of its production until the last day of storage before consumption for up to 90 days.

Introduction

Nitrosamines are formed by reactions between nitrite and structures containing amino groups. (Sanches Filho et al. 2003). Studies have reported that nitrosamines are also formed in environmental matrices such as plants, mud, and soil (Qiu et al. 2017). However, it is known that foods are the primary source of our exposure to nitrosamines (Gushgari & Halden 2018). In particular, the presence of nitrosamine in processed meat products is very important. Therefore, despite these important advantages of nitrite (Horsch 2013), which is used for antimicrobial agent and color development in meat and meat products (Park et al. 2015), it has become necessary to reduce its amount or eliminate it completely, especially in heat-treated meat products (Marriott et al. 1981). However, since there is no better additive that can be used instead, its limited use continues within the legal regulations. In this context, the production of such processed foods should be carried out with care in terms of the amount of nitrite added and the processes used because it is of great importance for public health (Ozbay et al. 2019) because it has been experimentally proved that some nitrosamines cause cancer (Wilkens et al. 1996).

Processed meat products are important in terms of nitrosamine formation, which is toxic to human health from the first to the last stage of production. Many factors affect the formation of N-nitrosamine in processed meat products. Among these factors, there are parameters such as different temperatures, heat treatment times, technological differences applied, packaging, additives used, as well as the nitrate, nitrite, primary, secondary and tertiary amines, amides, proteins, peptides, amino acids, different precursors and microbial activity also required (Yurchenko and Mölder 2007). On the other hand, the amount of N-nitrosamine in meat products changes depending on the presence of any inhibitors (ascorbic acid, tocopherols, etc.) in the environment (Fiddler et al. 1978), the residual or added nitrite concentration and the storage conditions (Yurchenko and Mölder 2007). For example, it is known that the presence of biogenic amines such as putrescine and cadaverine in the environment increases the formation of nitrosamine (Li et al. 2013), while ascorbic acid decreases it (Sun et al. 2016).

Regarding nitrosaminies, the possible carcinogenic effect of VNA prominent in the statements of institutions such as WHO (World Health Organization) and EPA (United States Environmental Protection Agency), has also been confirmed by IARC (The International Agency for Research on Cancer). It is known that some volatile derivatives (NDMA, NDBA, NDPA, NMEA, NDEA, NPYR, NPIP) (IARC 1987) may pose a particular risk in processed meat products. This study determined the amounts of VNA derivatives in commercial sausage formulations produced in Turkey and the effect of adding ascorbic acid to these formulations. Thus, the study aimed to determine the levels of these VNA in each of the production steps, at different packages (Vacuum and MAP- (Modified atmosphere packaging)) and during the storage process.

Material and method

Material

In this study, samples of sausage product at different production stages produced experimentally at Ankara Meat and Milk Board Sincan Meat Complex facilities were used as research materials. Sausages were produced by adding a fixed amount of nitrite salt (0.4%) to the sausage dough in all production processes. Experimental sausage production was done in two different ways: either, ascorbic acid (aa) was added at the level (500 ppm) of commercial production, or at twice the level (1000 ppm) of the commercial sausage formulation. Each experimental production was carried out in triplicate.

Samples were taken after each heat treatment in experimental production. A total of 36 samples of 100–120 g sealed in sample bags were used as the test materials. Samples were stored at 4 °C until analysis of VNA.

As the production was completed, the produced sausages were packaged in two different packages (MAP and vacuum). Packaged samples were collected to examine the impact of the storage process and packaging. For this purpose, a total of 40 packaged samples with 20 MAP and 20 vacuum packaging were taken in 200 g packages. The samples were stored in their packaging at 4 °C until they were analyzed.

Determination of N-nitrosamines

All taken samples were extracted for GC–MS analysis. The extraction method was based on that developed by Yuan et al. (2015). Briefly 20 g homogenized sample was taken and left in an ultrasonic water bath with 40 ml of DClM (Dichloromethane) for 15 min, and filtered with filter paper, then the filtrate collected. The same process was repeated. Then the solvent was evaporated in the rotary evaporator. The solvent-removed extract was collected with 1 ml of methanol, filtered, and placed in glass vials. Samples were vortexed and analyzed in GC–MS.

GC/MS (Agilent Technologies, 7890A / Agilent Technologies, 5975C, Santa Clara, CA, USA) was used for the detection of nitrosamines. Helium, as carrier gas and DB624 (30 m, 0.25 mm I.D and 1.40 μm, Agilent, St Louis, MO, USA) as column were used in the system, and it was operated in SIM mode. The oven was programmed as follows: 60 °C held for 2 min, ramp to 120 °C at 20 °C/min, held for 1 min, and then ramped to 220 °C at 20 °C/min, held for 2 min. Nitrosamine standard (EPA 521 Nitrosamine Mix, 2000 ppm, Sigma-Aldrich,USA) was used for identification. Retention times, molecular weight, qualifier ions for the detection in MS SIM are shown in Table 1.

Table 1.

Retention times, molecular weight,qualifier ions for the detection in MS SIM mode applied method for the 7 NAs using GC–MS

Compound	Weight (M/W)	Rt (min)	Target ions	Qualifier ions
NDMA	74	5.441	74.1	84.0/86.0
NMEA	88	6.82	88.1	71.1/73.1
NDEA	102	7.839	102.1	71.1/73.1
NDPA	130	10.071	70.1	113.1/130.1
NPYR	100	10.426	100.1	71.1/85.1
NPIP	114	10.909	114.1	71.1/85.1
NDBA	158	12.664	84.1	99.1/116.1

N-Nitrosodimethylamine (NDMA, R² = 0.983030, LOD = 0.11 μg/kg, LOQ = 0.36 μg/kg), N-Nitrosomethyletylamine (NMEA, R² = 0.972287, LOD = 0.12 μg/kg, LOQ = 0.40), N-Nitrosodiethylamine (NDEA, R² = 0.981711, LOD = 0.11 μg/kg, LOQ = 0.37 μg/kg), N-Nitrosopropylamine (NDPA, R² = 0.979911, LOD = 0.10 μg/kg, LOQ = 0.32 μg/kg), N-nitrosopyrrolidine (NPYR, R² = 0.983214, LOD = 0.06 μg/kg, LOQ = 0.21 μg/kg), N-Nitrosopiperidine (NPIP, R² = 0.989461, LOD = 0.17 μg/kg, LOQ = 0.56 μg/kg) and N- Nitrosodibutylamine (NDBA, R² = 0.967297, LOD = 0.08 μg/kg, LOQ = 0.26 μg/kg) separated and determined. The results were given as μg/kg. Method validation results are shown in Table 2.

Table 2.

Method validation results

Compounds	Calibration (µg/L)	Linearity (r2)	LOD (µg/kg)	LOQ (µg/kg)	Recovery (%)	Reproducibility (%RSD)
NDMA	0,5—75	0.999225	0.11	0.36	87.70	3.5342
NMEA	0,5—75	0.989719	0.12	0.40	92.90	2.0331
NDEA	0,5—75	0.998683	0.11	0.37	90.02	8.1090
NDPA	0,5—75	0.979911	0.10	0.32	87.44	5.0704
NPYR	0,5—75	0.999627	0.06	0.21	94.5	3.4497
NPIP	0,5—75	0.995153	0.17	0.56	93.9	4.5262
NDBA	0,5—75	0.994560	0.08	0.26	95.82	5.0957

Statistical analysis

The experimental design was 2 × 2 × 6 factorial in a completely randomized design with the addition of ascorbic acid (500 ppm and 1000 ppm), the material of packaging (vacuum and MAP) and storage time (0, 7, 15, 30, 60, 90 d). Data were evaluated by multivariate test using a general linear model (GLM) considering the factors and their interactions as fixed effects and the replicates as a random effect. Where factors and interactions were significant, differences between means were determined using the Duncan's multiple range tests at the P < 0.05 level. For each type of sausage dough, all experiments were carried out in triplicate. All statistical analyses were performed using the SPSS version 24 statistical program (IBM Corp., Armonk, NY, USA).

Result and discussion

According to the results of the research, no VNA derivatives were detected in any of the samples taken in the first four production stages in both forms of experimental commercial sausage production (500 ppm aa/1000 ppm aa). Following the 5th heat treatment stage of the production, VNA derivatives were found in both production formulations VNA and their amounts (ppb) are shown in Fig. 1.

Fig. 1.

N-nitrosamine levels (ppb) in sausage at the end of the production line

Following the final heat treatment, the sausages were packaged in two different packaging forms. These sausages have been analyzed to understand the effect of the packaging and storage process. Accordingly, as the process progresses, it was observed that the amount of N-nitrosamine increased and the packaging type also affected this formation. The amounts of N-nitrosamine formed during the storage period are shown in Fig. 2 and Fig. 3. VNA derivatives and their amounts (ppb) are shown in Fig. 1.

Fig. 2.

Volatile N-nitrosamine level (ppb) during the storage process of the sausage in vacuum package

Fig. 3.

Volatile N-nitrosamine level (ppb) during the storage process of the sausage in the MAP package

The statistical analysis of the effects of ascorbic acid used in production, storage process and packaging type on the VNA level is shown in Table 3.

Table 3.

The effect of ascorbic acid on N-nitrosamine formation in sausage

Variance Source	N	NDMA ppb	NMEA ppb	NDEA ppb	NDPA ppb	NPYR ppb	NPIP ppb	NDBA ppb
Production Line (PL)
1000 ppm	36	< LOQ	0.08	0.02	0.08	< LOQ	< LOQ	< LOQ
500 ppm	36	< LOQ	0.42	0.25	0.07	< LOQ	0.12	0.08
Significance		–	*	*	NS	–	*	*
Packaging Type (PT)
Vacuum	72	< LOQ	5.12	7.05	1.56	1.63	16.95	2.62
MAP	72	0.51	4.75	3.43	0.85	12.4	11.53	1.37
Significance		*	NS	*	*	*	*	*

^*P  < .05, NS not significant

As seen in Table 3, the change of ascorbic acid level during the production process had a significant effect on the amount of all N-nitrosamine derivatives except NDPA (P < 0.05). The difference in packaging types produced a significant difference in all derivatives except NMEA (P > 0.05). During the storage process, as time progressed, VNA levels increased substantially(Table 4).

Table 4.

Effect of ascorbic acid on nitrosamine formation in sausage

Variance Source	N	NDMA ppb	NMEA ppb	NDEA ppb	NDPA ppb	NPYR ppb	NPIP ppb	NDBA ppb
Storage Period (day) SP
0	24	< LOQ	1.64	0.95	0.52	0.67	1.27	< LOQ
7	24	< LOQ	1.24	2.46	0.54	0.94	12.56	1.20
15	24	< LOQ	1.76	2.77	0.67	5.61	18.05	1.33
30	24	0.33	1.75	2.87	0.83	7.52	13.9	1.53
60	24	0.38	6.65	7.56	1.03	10.02	13.0	2.97
90	24	0.57	13.26	10.51	2.97	10.98	13.62	3.10
Significance		*	*	*	*	*	*	*
PT * SP		*	*	*	*	*	*	*
PT * PL		*	NS	*	*	*	NS	*
SP * PL		*	*	*	*	*	NS	*
PT * SP * PL		*	NS	*	*	*	*	*

^*P  <  .05, NS not significant, PT Packaging Type, SP Storage Period, PL Production Line

According to the findings, VNA formation was not detected in the sausage dough and in the first four heat treatment processes of the production (< LOQ). However, it was observed that NMEA, NDEA, NDPA, NPIP and NDBA derivatives were formed with the fifth (final) heat treatment. Among the VNA derivatives which formed following the last step of the traditional production process (500 ppm aa), NMEA was formed at the highest level with 2.53 ppb. In the sausages produced with the second formulation (1000 ppm aa), at this stage NMEA was formed at the level of 0.47 ppb. This is due to the fact that the nitrite salt, added to the sausage at the beginning, reacts with the amine structures in the meat by heat treatments in the production process and forms VNA derivatives (Sanches Filho et al. 2003). Campillo et al. (2011) have also emphasized that not only cured products, but also burgers, meatballs, or raw products that have not been heat-treated may contain VNA.

At the end of the production, the average total VNA amount was determined at the level of 5.62 ppb in the packaged sausage sample which produced by the traditional method (500 ppm aa). Byun et al. (2004) reported that the total VNA level of commercial meat products in the United States of America was around 10 ppb. Jo et al. (2003) measured this amount at the level of 10—40 ppb for sausages. An analysis of VNA levels of processed meat products in South Korea reported their presence at 36.8 ppb (Byun et al. 2004). Ozel et al. (2010) determined the total VNA concentrations of 22 samples containing sausage, salami, soudjouk and doner kebab at 0.45–16.63 ppb levels. Researchers emphasized that NDMA and NDEA derivatives are generally found in meat products. Rywotycki et al. (2003) reported that animal species, animal nutrition types and even seasonal effects directly affect the formation of VNA in raw meat. Lee (2019) reported that the total amount of VNA in sausages varies between 2—11 ppb and that NDMA, NPIP and NPYR occur mostly in sausages. In another study, Cintya et al. (2019) encountered non-volatile N-nitrosamine derivatives such as N-nitroso-thiazolidine-4- carboxylic acid (NTCA) and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA) in 20 different samples containing sausage, smoked meat, burgers and canned meat at high levels. While the amount of NTCA in the samples varied between 500–4227 ppb, the amount of NMTCA varied between 20–990 ppb. However, the researchers could not detect any volatile NDEA derivative in any of their products. Campillo et al. (2011) reported that the most common VNA derivatives in processed meat products are NDMA and NPIP. In these variable data, mostly, the lack of information about the storage method and duration of the samples which collected during the survey studies is the biggest factor. At the same time, not controlling the packaging, production method and content that will affect the formation of nitrosamine also affects the results.

According to the storage period findings of this study, NDMA did not occur until the end of the storage period under vacuum packaging. However, it was observed that the NDMA in the samples packaged with MAP packaging increased (0.92 ppb) following the 30th day and it formed at the highest level (1.20 ppb) at the last day of storage. It was also found that other VNA derivatives increasingly occurred in general in both packages starting from the first week.

The formation curves for NPYR and NPIP were similar for both packaging types. However, it was determined that while NPYR formed at the level of 1.23 ppb with traditional production (500 ppm aa) at the end of the first week in the MAP packaging, the amount of NPYR reached the level of 2.22 ppb at the last day of storage. This formation, when the amount of ascorbic acid increased (1000 ppm aa), was 0.48 ppb at the beginning and reached 1.88 ppb on the 90th day of storage. De Mey et al. (2014) reported that they detected carcinogenic VNA derivatives at the end of their shelf life in sausages sold in Belgian markets.

Formation of another VNA analyzed, NPIP was at similar levels in both types of packaging. Researchers have reported in a different study that especially the product named "Pepper Salami" contains 12,3 ppb NPIP at the end of its shelf life. Since black pepper and red pepper are precursors for NPYR and NPIP formation, it increases the formation amount especially in peppery products. (Lee 2019).

According to the findings another study, ascorbic acid provided an advantage in the formation of VNA during the storage period. Li et al. (2012) reported that the use of ascorbic acid in the ripening process of dry cured sausage reduces the VNA level. They also emphasized that NDMA, NDEA and NPYR are formed thoughout the ripening process. In a different study, the relationship between Lactobacillus pentosus which was inoculated into dry fermented sausage during the ripening process and VNA was determined. This VNA formation arises from the chemical and microbiological reactions in the process (Xiao et al. 2018). In particular, microorganisms can cause N-nitrosamine formation by reducing nitrate to nitrite and breaking down the proteins into amines and amino acids (Tricker and Preussmann 1991).

Regarding NDBA, another VNA derivative, an increase was observed in both packaging types over time. It was determined that at the end of the first week NDBA did not occur in both types of production in MAP packaging but on the last day of the storage, NDBA occurred at the level of 2.79 ppb in traditional production (500 ppm aa) and at the level of 2.57 ppb in the second type of production (1000 ppm aa). In vacuum packaging, while NDBA started to occur from the first week, the amount remained at a similar level during the storage, and it was measured at 3.54 ppb with traditional production on the last day of the storage and 3.36 ppb with the second type of production.

When all packaging process data are evaluated together, it is possible to say that VNA levels increase in both vacuum and MAP packaging systems as the storage time progresses. Precursor accumulation, chemical and / or microbiological reactions, depending on the environmental conditions, can be seen as the main reason for this formation (Xiao et al. 2018). In the study, MAP packaging atmosphere content was determined as 30% CO2 and 70% N2 as part of the production process. In this way, by providing anaerobic conditions under the MAP packaging, even if aerobic bacteria are prevented from proliferation, it will take time to completely eliminate them (Tayar & Hecer 2013). In this respect, it is thought that the microbiological activity that continues in the MAP packaging, albeit limited, increases the formation of VNA.

In another study with shorter storage periods, Domanska-Blicharz et al. (2004) performed NDMA, NDEA, NDPA, NDBA, NPIP, NPYR and NMOR analyses in processed meat products stored in the refrigerator for 72 h and they found NDMA at 2.6–2.8 ppb levels. In this study, while NDMA under both vacuum and MAP packaging and NDBA in MAP packaging could not be detected in our sausages that were stored for seven days, all other investigated VNA derivatives formed.

The effect of ascorbic acid on all processes is also of great importance in terms of the results of the research. It is emphasized that ascorbic acid helps to decrease the VNA level and in particular, it reduces the formation of NDMA (Li et al 2012; Marriott et al. 1981; Mejborn et al. 2019; Pirincci et al. 1986 and Rywotycki 2002). This is due to the fact that ascorbic acid decreases the nitrite amount gradually by reducing the nitrite to nitric oxide (Li et al. 2013). Similarly, Rywotychi (2002) emphasized that in their study they found that NaCl and sodium ascorbate significantly reduced the amount of NDMA and NDEA in the pork sample.

In the light of all the data obtained, it is possible to say that the possible carcinogenic VNA derivative occurred in the sausage from production to the last day of storage. It is also seen that the second sausage formulation (1000 ppm aa) significantly reduces this amount for each stage (P < 0.05). The increase in the amount of VNA formed in the processed meat product during the storage process agrees with other studies (Domanska-Blicharz et al. 2004 and Xiao et al. 2018).

The study shows that the formation of VNA can be greatly affected by environmental conditions. Storage, packaging method, storage time variables significantly affect VNA levels. Different studies have also indicated the variability of VNA levels.

Pirinçi et al. (1986), in their research on sausage, obtained total VNA values ranging from 1.4 to 600 ppb for different VNA derivatives. This study found that NPYR was found in all samples, while NDMA, NDEA and NPIP were found in 95% of the samples. Gushgari and Halden (2018) reported in their review study that 118 different processed meat products contained total VNA at levels between 0.1 and 121 ppb.

In today's conditions, production conditions have been improved, the amount of additives have been under more strict control and VNA levels have been reduced as much as possible. However, it should not be forgotten that the complex structure of the VNA formation mechanism may produce highly variable VNA derivatives and amounts under different production and consumption conditions.

As a result, the sausage product carries a risk in terms of VNA formation from its production to the last day of its storage. Increasing the amount of ascorbic acid during production helped to reduce the total VNA level at each stage. In addition, consuming packaged sausages by considering the production date can prevent the intake of nitrosamines that may form during the shelf life. It is also known that healthy choices in food consumption will significantly reduce VNA intake (Gushgari & Halden 2018). The results show that ascorbic acid significantly reduces VNA formation. In addition, the study shows that VNA formation increases during the storage period and that risks arise in both types of packaging. In this respect, the rapid consumption of such processed meat products following production can minimize VNA consumption. Despite the advantage of increasing the amount of ascorbic acid, preliminary tests should be carried out, especially with panel tests.

Abbreviations

aa

Ascorbic acid

DClM

Dichloromethane

EPA

United States Environmental Protection Agency

GC–MS

Gas chromatography–mass spectrometry

GLM

General linear model

IARC

The International Agency for Research on Cancer

MAP

Modified atmosphere packaging

N

Sample size

NDBA

N- Nitrosodibutylamine

NDEA

N-Nitrosodiethylamine

NDMA

N-Nitrosodimethylamine

NDPA

N-Nitrosopropylamine

NMEA

N-Nitrosomethylethylamine

NPIP

N-Nitrosopiperidine

NPYR

N-nitrosopyrrolidine

R²

R-squared

VNA

Volatile N-nitrosamine

WHO

World Health Organization

Authors' contributions

SÖ (Research/Analysis/Writing).

UTŞ (Supervisor).

Funding

Ankara University Coordinating Office for Scientific Research Projects (Ankara, TURKEY) Project Number: 17L0239017.

Availability of data and material

Data transparently transferred completely and accurately.

Declarations

Conflicts of interest

The authors declare that they do not have any conflicts of interest.

Ethics approval (include appropriate approvals or waivers)

This research does not need ethical approval.

Consent to participate

All authors of the article accepted that the manuscript (sent to your journal) contains this information.

Consent for publication

Enclosed is the electronic format of the manuscript by Sena ÖZBAY and U. Tansel ŞİRELİ, entitled “The Effect of Ascorbic Acid, Storage Period and Packaging Material on the Formation of Volatile N-Nitrosamine in Sausages” which is being submitted for possible publication in “Journal of Food Science and Technology”. Our paper article is original and unpublished research.