Currently, meat products are one of the most consumed foods in the world. In the United States and most European countries, meat is one of the costliest foods. Meat accounts for 35% of household expenses in Denmark, France, and Belgium; 30% in Italy, Spain, and Ireland; and 25% in the UK and the Netherlands (Jiménez-Colmenero
et al., 2001). Thus, production of meat products is developing rapidly in the world since they are more affordable than the ordinary fresh meat. In this regard, 40% red meat sausage is one of these products, which can partly eliminate the need for animal proteins (Hashemi and Shokraneh, 2013). Meat products contain nitrite that is a key ingredient in the processed meat products. Nitrite has different functions (NANO2) in meat products. The initial application of nitrite is to create and develop a special taste of meat products and prevents bad smells caused by oxidation of
lipids (Esmaeilzadeh
et al., 2012). In the next step, nitrite reacts with meat myoglobin and produces
a special pink color with the production of nitrosohemochrome. Nitrite can also prevent the growth of pathogenic bacteria, especially clostridium species. Despite the benefits mentioned above, high nitrite levels in meat products are harmful to health. Furthermore, Nitro acid may be produced as a result of nitro oxide hydration by sodium nitrite reduction and turn to N-nitroso compounds especially nitrosamines in reaction with type 2 amines and amino acids in the muscle. These compounds are remarkable for their carcinogenicity. Such negative potential effects increase the tendency to replace and reduce the contribution of nitrite to meat products (Toldrá
et al., 2009). Based on the standard limit for meat products, the limit for nitrites should not exceed 500 ppm for meat products (60%) (Al-Shuibi and Al-Abdullah, 2002). Although reduction of nitrite in meat emulsions is desirable for the aforementioned negative effects, it promotes the lipid oxidation reaction. The compounds produced by the oxidation reaction are capable of reacting with oxygen at high speed. The reaction speed can be delayed by adding antioxidants. Types of synthetic antioxidants, such as Tertiary butyl hydroquinone, Butyl hydroxyl anisole, and Butyl hydroxyl toluene are used in the food industry to prevent lipid oxidation, but their use in the industry is limited by the potential for health and toxicity (Domínguez
et al., 2019). There is an increasing demand in consumers to use natural additives as a substitute and preservative factor in foods during the recent years due to their safety in comparison to synthetic additives. Some of these compounds are extracted from plants or some of the pigments, particularly in some microbial, animal or plant pigments known as safe compounds (Stchigel
et al., 2004). From the production point of view, microbial pigments are economical and inexpensive and their extraction is simple. Moreover, if an appropriate strain is selected for extraction, the microbial pigments can have a high yield. In terms of raw materials, no shortage was observed in the production of microbial pigments. Moreover, seasonal changes do not have any impact on their production process. Regarding safety and health, microbial pigments do not pose a risk on the human. Some microbial pigments can be useful since they can have antioxidant, anti-cancer, antimicrobial, and anti-inflammatory effects. They also can be vitamin precursors along with coloring the food. Microbial pigments can be extracted from bacteria, molds, yeasts, as well as seaweed and sea molds (Nazemi
et al., 2011). Microbial pigments are used in the food industry for processing all kinds of foods. One of these important pigments is Anka's mold pigment called
Monascus, which is a red intracellular pigment. Application of this fungus has long been used in eastern countries to produce color (Erdoğrul and Azirak, 2004). The meaning of the term in ancient Chinese is
Monascus purpureus, i.e., red yeast rice. The red pigment is important in industry
and especially in the meat industry, because it
can replace the illegal artificial colors (Akihisa
et al., 2005). Many researches indicated that
Monascus pigments exhibit biological activities, like anticancer, antihyperlipidemic, and anti-inflammatory activities (Hong
et al., 2008). According to (Wójciak
et al., 2019), intake of 200 ppm of Khuzestani sativa in the 60% meat Frankfurter sausage formulation has a favorable effect on the prevention of lipid oxidation, similar to that of 500 ppm sodium nitrite. Considering the disadvantages of nitrate and the advantages of
Monascus purpureus Pigment, the overall aim of this study was to investigate the possibility of replacing sodium nitrite with
Monascus purpureus pigment in 40% meat German sausage and to assess its antimicrobial, antioxidant, color, and sensory properties.
Materials and Methods
The
Monascus purpureus purpureus CMU001 pigment was prepared from the mycology group of Isfahan University of Medical Sciences. Calf meat was purchased from Brazil. The starch was prepared from Fara-Daneh Inc. (Shiraz, Iran). The spices, sugar, salt, and wheat flour were purchased from Golestan company, Iran. Sodium nitrite was purchased from shanghai chemex group ltd, China. Phosphate and ascorbic acid were purchased from Sigma-Aldrich (U.S.A). All chemicals and microbials materials needed to perform tests were bought from Merck Company, Germany.
Sample preparation: The
Monascus purpureus pigment was prepared by alkali method using the Haghparast method (Sharmila
et al., 2013). By this method, the
Monuscus pigment was extracted from the powdered pericarp by shaking in a 0.13 molar solution of potassium hydroxide. Later, the filtering was done to remove impurities, pigments were precipitated with hydrochloric acid, and the solution was filtered and dried. To remove bad odors, dried sediments were washed with petroleum ether. At the end, pigments were mixed with alkali and dissolved in water.
The treatments were prepared according to the usual method of producing 40% meat sausage in Pardis factory by replacing 0, 20, 40, 60, 80, and 100% nitrite with
Monascus purpureus pigment. The frozen meat was ground. The ground meat entered the Laska cutter. In the next step, ice was added to the cutter with
Monascus purpureus pigment, nitrite, spices, salt, and phosphate. The cauterization process continued for 2 to 3 minutes. Later, the oil was added and after several rounds (3 minutes) of cauterization, filler materials (wheat flour and starch) and other additives (sugar and ascorbic acid) were added and the process was completed for 4 to 5 minutes until a fully emulsified paste was created with a temperature of 4 to 5 degrees celsius. The dough was entered to the Wolfkin filler and filled with 24 caliber polyamide coatings. The product was transferred to the baking room for thermal processing. After baking, carried out at 80 °C for a period of 45 minutes, the samples’ temperature were reduced by cold water showers and the samples were kept in a refrigerator at 4°C for 1, 10, 20, and 30 days for analysis (Hashemi and Shokraneh, 2013).
Thiobarbituric acid (TBA): It was measured according to a previous study (Ulu, 2004).The total counting of microorganisms was carried out according (Sharifi-Yazdi
et al., 2016) using the Plate Count Agar (PCA) culture medium. The mold and yeast were measured according to (Maktabi
et al., 2016) using Dichloran Rose Bengal Chloramphenicol (DRBC) medium.
Staphylococcus aureus was measured according to (Javadi and Safarmashaei, 2011). To this end, three factors of
a*,
b*, and
L* were measured by color measurement model CR-400 Minolta, Japan (Yam and Papadakis, 2004). The sensory properties of the samples were evaluated based on the 5-point hedonic method and using 10 trained evaluators to assess overall acceptability of the samples (Yam and Papadakis, 2004).
Data analysis: The results of the tests were evaluated in three replications. To compare the mean of data, Duncan's one-way analysis of variance was used at 95% confidence level by Minitab 16 software as well as Excel software to plot the graphs.
Results
Analyzing antioxidant properties of the produced sausage samples: According to
Table 1, no significant difference was found between the amount of thiobarbituric acid in all treatments on the first day of storage (
P ≤ 0.05). The amount of thiobarbituric acid during the storage period was increased in all treatments. In other words, the highest amount of thiobarbituric acid (2.68 mg malondialdehyde per kg) after 30 days of storage belonged to the treatment containing 100%
Monascus purpureus pigment. However, the lowest amount of thiobarbituric acid (2 mg malonaldehyde per kg) belonged to the treatment containing 100% nitrite (control), which had a significant difference with each other. Thiobarbituric acid content was increased in all treatments after increasing the content of
Monascus purpureus pigment and decreasing nitrite content, but this trend was not significant by replacing 60% nitrite with
Monascus purpureus pigment (
P ≤ 0.05).
The microbial properties of samples of sausages produced: According to
Figure 1, the total count of bacteria,
staphylococcus aureus, molds, yeasts, and coliforms had an increasing trend in all treatments during the storage period. So, the lowest total count of bacteria,
staphylococcus aureus, molds and yeasts, as well as coliforms were observed among treatments in samples containing 100% sodium nitrite.
Evaluating the color properties of produced sausages L* index: The results of
Figure 2 indicate the effect of different concentrations of
Monascus purpureus pigments on the mean of
L* index. According to the findings, application of
Monascus purpureus pigment instead of nitrite used in sausage formulation and an increase in its concentration caused a slight decrease in
L* index compared to the control sample. However, this reduction was not statistically significant up to 60% replacement concentration of
Monascus purpureus pigment instead of nitrite. A significant difference was observed between the levels of
L* between the control sample and treatments containing 80% and 100%
Monascus purpureus pigments.
Figures 3 illustrates the effects of using different amounts of
Monascus purpureus pigment instead of nitrite on changes in
a* index (red color) and
b* index (yellow color) in the formulation of German sausages. The results showed that by increasing the concentration of
Monascus purpureus pigment instead of nitrite in the formulation of German sausages, the
a* and
b* index increased significantly. In other words, the highest rates of
a* and
b* indices were observed in the sample containing 100%
Monascus purpureus pigment, which had a significant difference with the control sample (100% nitrite). According to the results,
a* index had a slight increase during the storage period in all treatments, which was not statistically significant. The
b* index had a slight decrease during the storage period in all treatments, which was not statistically significant.
Sensory evaluation (Overall acceptance): Figure 4 represents results of the overall acceptance of sausage samples containing different concentrations of
Monascus purpureus pigment instead of nitrite. The overall acceptance score of sausage samples containing different concentrations of
Monascus purpureus pigment was slightly higher than the control sample, which was not statistically significant (
P ≤ 0.05). Therefore, no significant difference was observed between the overall acceptance score of samples containing different concentrations of
Monascus purpureus pigment and the control sample. The results indicated that the overall acceptance score of the treatments showed a slight increase or decrease during the storage period, in which these changes were not statistically significant (
P > 0.05) compared to the first day of production.