Mechanically separated meat (MSM) is widely used in the meat industry and is one of the cheapest animal origin materials [1
]. In recent years, Poland has become one of the biggest poultry meat producers in Europe [2
]. Mechanically separated poultry meat (MSPM) production can fully exploit soft tissue residues from the bones and poultry carcasses remaining after hand deboning [1
The most commonly used separators in MSM production are high-pressure ones, low-pressure ones are used less commonly [3
]. The risk of higher microorganism levels in the product increases together with the increased grinding level and increased muscle fiber degradation. Thus, MSPM obtained with the high-pressure method is a good substrate for microflora growth [3
]. Excessive fragmentation of MSPM, decay of tissue structure and aeration during the separation process fosters microbiological pollution. What is more, the raw material for MSPM production might contain a relatively high number of microorganisms. In the MSM microflora, the following pathogenic may be present in the bacteria: Salmonella
spp., Escherichia coli
, Listeria monocytogenes
, Yersinia enterocolitica
, Staphylococcus aureus
, as well as saprophytic Pseudomonas
, that speed up meat spoilage [4
]. A substantial factor determining MSPM microbiological quality is the hygiene of carcass processing as well as the level and type of waste at the early production stage and during animal rearing [9
]. MSPM is usually affected with microorganisms originating from chicken carcasses and bones as well as from the environment in which it is processed (service, equipment, and environment). Additionally, if the temperature during the deboning process is not low enough, the risk of bacteria multiplying increases [10
]. The presence of many bacteria genus, including the pathogenic ones like Enterobacteriaceae and Salmonella
in the raw material of poultry is well documented [7
Due to low microbiological MSPM stability it should be sent for processing immediately after production or should be chilled to a temperature of 2 °C. MSPM after chilling may be stored but not longer than 24 h, which significantly hinders the logistics of processing the MSPM in the plant.
One of the methods which enables the lengthening of MSPM shelf-life is to apply the freezing process to −18 °C [14
]. Nevertheless, this process causes many negative changes within MSPM i.e., a worse water holding capacity, drip loss, color change, lower protein solubility and other changes [15
]. In the industry the practice of curing fresh or defrosted MSPM with sodium nitrite is also applied. Considering the proven negative effect of nitrites and especially their derivates N-nitrosamines (NAs) on human health it is recommended to reduce their level in the diet [16
]. Research has indicated that NAs formation depends on the level of nitrites added to meat [17
]. In some countries e.g., in Denmark internal regulations were introduced to lower the maximum level of nitrites from 150 mg/kg allowed in the European Union to 60 mg/kg [19
]. Reducing the amount of nitrites in cured MSPM batters may have an influence on their microbiological quality and thus have a negative effect on shelf-life and the hygienic security of products with MSPM content. Thus, it is important to look for new technological solutions that will constitute an additional barrier in the ‘hurdle technology’ in producing meat products with MSPM and lower sodium nitrite levels.
Lactic acid bacteria (LAB) plays an important role in the production of fermented meat products, they affect the taste and texture as well as extend the shelf-life of these products. They also may assure meat safety without any negative effects on product quality [20
]. Lactic acid bacteria—i.e., Lactobacillus sakei
, Pediococcus acidilactici
, Lactobacillus curvatus
—are used in order to increase raw meat products shelf-life and health safety [22
]. The antimicrobial LAB activity is attributed to the metabolites produced by the bacteria cell. Apart from organic acids, hydrogen peroxide, diacetyl, and CO2
, they also produce an abundant group of protein substances of significant antimicrobial and/or bacteriostatic activity called bacteriocins [23
]. In the research of Łaszkiewicz et al. [24
], it was proved that the Lactiplantibacillus plantarum
SCH1 strain isolated from ecological raw fermented pork roast it is possible for it to be used to inhibit E. coli
growth in raw uncured MSPM batter after 4 days of cold storage and also in raw MSPM batters cured with a lower amount of sodium nitrite (50 mg/kg) during storage. The results reported by other the authors indicate that some LAB strains are thermotolerant and thus could be used to elongate the shelf-life of cooked meat products [26
]. In this research, the L. plantarum
SCH1 strain was used in MSPM batter cured with a lower amount of sodium nitrite and next the model cooked meat product was formed for assessment.
The aim of the research was to assess the possibility of using Lactiplantibacillus plantarum SCH1 for the bioconservation of cooked sausage made from mechanically separated poultry meat cured with a reduced amount of sodium nitrite.
2. Materials and Methods
2.1. Bacteria Culture and Growth Condition
The Lactiplantibacillus plantarum
SCH1 strain isolated from ecological raw fermented pork roast was used within the research. The strain originated from a microorganisms collection owned by the Facility of Food Hygiene and Quality Management, Warsaw University of Life Sciences in Poland and was chosen based on the research of Rzepkowska et al. [28
] and Łaszkiewicz et al. [24
]. Lactic bacteria cultures stored in 20% glycerol at −80 °C were defrosted to prepare the strain for the research. The 20 µL of the strain after mixing was put into 5 mL of MRS broth (Merck KGaA, Darmstadt, Germany) in sterile test tubes and incubated at 37 °C for 24 h. After the next decimal dilution and incubation in the same conditions the cells were centrifuged in a J2-21 centrifuge (Beckman, Birkerød, Denmark) at 4500 RPM (2313× g
). Then, after removing supernatant bacteria cells, it was suspended in a NaCl solution (0.9%) and applied into the MSPM batter to reach the bacteria concentration of approx. 107
cfu/g. All of the procedures were performed aseptically using sterile equipment.
2.2. Preparing Batters and Producing Sausages with MSPM
High pressure MSPM was obtained in industrial conditions (diameter of the holes was 1 mm). Non-frozen chicken carcasses were deboned using the separator (AM2C, Quimper, France) and after separation the obtained MSPM was divided into blocks of 10 kg each and frozen to −18 °C. The blocks of MSPM were stored frozen for 2–4 days. For producing the sausage batters, the blocks of MSPM were defrosted in refrigerated conditions (4–6 °C) for approx. 24 h. Two MSPM batter treatments were prepared for sausage production: C—control treatment produced with MSPM cured with 50 mg/kg of sodium nitrite, L—treatment produced with MSPM cured with 50 mg/kg of sodium nitrite with the addition of Lactiplantibacillus plantarum SCH1 at about 107 cfu/g.
In the L treatment, the bacterial biomass suspended in the physiological saline (1.25 kg) was added to 10 kg of defrosted MSPM. In the C treatment, physiological saline (1.25 kg) without bacteria was added to the 10 kg of MSPM. Next, the MSPM (C and L) were mixed separately in the mixer (N-50G, Hobart Corporation, Troy, OH, USA) for 1 min and left in polypropylene containers (48 L capacity) for 4 days in cold storage at a temperature of 4 °C. Time and storing conditions were established based on the preliminary research [24
]. Next, water/ice, sodium nitrite (50 mg/kg), spices, and additional substances (Table 1
) were added to the MSPM (C and L) and after that it was minced in a cutter K40 (Seydelmann, Stuttgart, Germany), reaching up to 10 °C in the batter. The MSPM batters were stuffed into the semi-permeable casings (DAT SCHAUB, Suchy Las, Poland). After stuffing, the sausages were allowed to settle for 45 min at a temperature of 20–25 °C.
After that the sausages were smoked with hot smoke 56–65 °C for 50 min and then steamed to 70–72 °C up to reaching a final internal sausage temperature of 70 °C. The final products were chilled with water to 15–20 °C, and next additionally chilled with cold air in the cold store to 4–6 °C.
The tests of the model sausages with MSPM were performed after thermal treatment and chilling (after production) as well as at 1 and 3 weeks of storage. The experimental production was done in the technical hall of the Department of Meat and Fat Technology, Institute of Agricultural and Food Biotechnology in Warsaw (Poland). The experiment described above was repeated three times (n = 3) in independent trials (replicates).
2.3. pH Measurement
The 10 g meat product samples were mixed with 50 mL distilled water and homogenized (14.000 RPM) for 1 min with an 800 W blender (MSM 66120, BSH Hausgeräte GmbH, Munich, Germany). To determine the pH value a digital pH-meter (Mettler Delta 350, Mettler Toledo, Schwerzenbach, Switzerland) with an automatic compensation of temperature and a glass-calomel In a Lab Cool electrode (Mettler Toledo, Greifensee, Switzerland) was used.
2.4. Oxidative-Reduction Potential (ORP) Measurement
To determine the ORP value the 10 g meat product samples were mixed with 50 mL distilled water and homogenized (14.000 RPM) for 1 min with an 800 W blender (MSM 66120, BSH Hausgeräte GmbH, Munich, Germany). The redox potential was performed using a digital pH-meter (Mettler Delta 350, Mettler Toledo, Schwerzenbach, Switzerland) equipped with an In Lab Redox Pro electrode (Mettler Toledo, Greifensee, Switzerland). The obtained results were calculated into the value of ORP in relation to the standard hydrogen electrode EH (mv). The ORP value of the reference electrode at a temperature of 20 °C—Eref = 207 mV was summed up with the value obtained with the equipment.
2.5. Determining the Total Heme Pigments Content and Nitrosylmyoglobin Concentration
The total heme pigments content and nitrosylmyoglobin concentration was performed using the Hornsey method [29
]. To determine the absorbance of the filtrates value, a U-2900 spectrophotometer (Hitachi, Tokyo, Japan) was used. The total heme pigment content was measured in hematin ppm and was calculated by multiplying the absorbance at 640 nm by 680. The nitrosylmyoglobin concentration (NO-Mb) content was calculated by multiplying the absorbance at 540 nm by 290 and was finally expressed in g/100 g according to the formula
2.6. Sodium Nitrite and Nitrate Content
The content of nitrites and nitrates was performed according to PN-EN 120414:2006 with an amendment of Siu and Henshall [30
]. The 10 g homogenized sausage sample was mixed with deionized water added to the volume of 50 mL into a volumetric flask and heated for 20 min. at a temperature between 70–80 °C. After cooling, deionized water was added to the sample to a volume of 100 mL and mixed. The supernatant was filtered using cellulose acetate (CA) syringe filters (Alfatec Technology, Zagreb, Croatia) with a pore size of 0.45 µm and after that the filtrate was used for the HPLC analysis.
For the extracts analysis an Agilent 1200 liquid chromatograph (Agilent Technologies, Waldbronn, Germany) with a UV detector, analytical column IonPac® AS11-HC 4 × 250 mm (Thermo Fisher Scientific, Sunnyvale, CA, USA) and pre-column AG11-HC 4 × 50 mm (Thermo Fisher Scientific, Sunnyvale, CA, USA) was used. Nitrate and nitrite were separated in isocratic conditions: 10 mmol/L sodium hydroxide (Chempur, Piekary Śląskie, Poland) for 20 min, then the column was washed with 50 mmol/L sodium hydroxide for 10 min, and equilibrated with 10 mmol/L sodium hydroxide for 5 min. The volume of the injection was 25 µL, the eluent flow-rate 1.5 mL/min and analytes were detected at 225 nm with UV detection.
The content of nitrite and nitrate anions was expressed in terms of the salts: NaNO2 and NaNO3 in mg/kg.
2.7. Determination of Color
To determine the color a spherical CR-300 spectrophotometer (Konica Minolta, Tokyo, Japan) with a measuring hole of 25.4 mm in diameter was used. The measurement was performed in a laboratory at a temperature of 24 °C ± 2 °C. Computed trio-chromatic coordinates were expressed in the CIE L* a* b* system, where L* means lightness, a* chromaticity from green to red color and b* chromaticity from blue to yellow color. For the measurement of the color, the standard CIE observer was used: 2°, illuminant D65, 8 mm measuring area and the white a tile standard (L* = 95.87, a* = −0.49, b* = 2.39) as a reference source was used. For each of the three replicate treatments (C, L) four measurements were carried out. The a*/b* ratio was also calculated [31
2.8. Microbiological Tests
The scope of the MSPM sausages microbiological test in the first research stage (after production) was as follows: total viable counts (TVC), lactic acid bacteria counts, Escherichia coli counts, Enterobacteriaceae counts, coagulase positive Staphylococcus count and Salmonella spp., and Campylobacter spp. presence in 25 g. The range and methodology of the microbiological tests are described below.
Samples of 10 g or 25 g were taken aseptically for quantity or quality lab measurements and after mixing the samples was suspended in 100 mL or in 250 mL of peptone water, respectively. Then, the decimal dilutions were used and bacteria growth on different culture media was performed.
To determine the presence of Salmonella spp. in 25 g the Müller–Kauffman’s medium with tetrathionate and novobiocin (Oxoid Ltd., Basingstoke, Hampshire, UK), medium according to Rappaport-Vassilliads with soya (RVS, Oxoid Ltd., Basingstoke, Hampshire, UK), xylose lysine deoxycholate medium (Oxoid Ltd., Basingstoke, Hampshire, UK), and chromogenic medium Rambach (RA, Merck KGaA, Darmstadt, Germany) were used (37 °C for 24 h).
To determine the presence of Campylobacter spp. in 25 g a rapid serological test Singlepath Campylobacter (Merck KGaA, Darmstadt, Germany) was used. For incubation the Bolton broth medium (Oxoid Ltd., Basingstoke, Hampshire, UK) in a microaerophilic atmosphere (5% O2, 10% CO2 and 85% N2) initially at 32 °C for 4 h was used and then at 41.5 °C for 44 h.
PCA (Plate Count Agar) medium (Oxoid Ltd., Basingstoke, Hampshire, UK) at 30 °C for 48 h was used to evaluate the number of the total viable counts (TVC).
For the mesophilic lactic acid bacteria counts and the determination of the MRS (Man Rogosa Sharpe) agar medium (Merck KGaA, Darmstadt, Germany) at 30 °C for 48 h was used.
Escherichia coli counts was determined using agar TBX (Tryptone Bile X-Glucuronide) medium (Oxoid Ltd., Basingstoke, Hampshire, UK) with incubation at 44 °C for 18–24 h.
To determine the Enterobacteriaceae counts VRBD (Violet Red Bile Glucose) agar medium (Merck KGaA, Darmstadt, Germany) at 37 °C for 24 h was used.
The positive coagulase Staphylococcus enumeration was performed on RPF (Rabbit Plasma Fibrinogen) medium (Oxoid Ltd., Basingstoke, Hampshire, UK) at 37 °C for 18–24 h.
The number of bacteria was expressed as log10 of colony forming units per gram of MSPM (log cfu/g). In the case of E. coli and coagulase positive Staphylococcus if their presence was not confirmed in the sausage after production and 1 week of storage they were not determined after 3 weeks of refrigerated storage. In the case of Salmonella spp. and Campylobacter spp., if their presence was not confirmed in the sausage after production they were not determined in the further research stages during refrigerated storage. The remaining microbiological tests were performed on the products after production and also after 1 and 3 weeks of storage at 4 °C.
2.9. Biochemical Identification of LAB
The sausage samples were inoculated on plates with MRS agar as described above. After incubation the cultures were taken randomly, 10 colonies per each sample and placed in the MRS broth (LabM, Heywood, UK). Identification of isolated bacteria strains whether or not they belonged to the Lactobacillus genus was confirmed by Grama staining and by observing cells under a microscope as well as by a catalase test. Species identification was performed using an API CHL50 test (bioMérieux Polska Sp. z o.o, Warsaw, Poland) according to the producers’ manual. Biochemical profiles obtained as carbohydrates fermentation results interpreted with an apiwebTM base allowed to qualify the tested isolates to the species. Next, they were compared to the L. plantarum SCH1 strain profile used within the experiment.
2.10. Sensory Assessment
The sensory assessment was performed using a Quantitative Descriptive Profile (QDP) method according to the ISO 13299:2016 norm. The assessing teams’ task was to determine the intensity of each of the mentioned quality traits and to put their own assessment on the adequate non-structured graphic scale (0–10). In order to compare the sensory quality of the model meat products in the sensory assessment after production, the following parameters were used: four taste discriminants (of cured meat, acidic, fatty, salty, where 0—the least intense, 10—the most intense), three flavor determinants (of cured meat, acidic/spicy, fatty, where 0—the least intense, 10—the most intense) also the following traits were estimated: flavor desirability, color tone, tenderness, juiciness, taste desirability, general quality, where 0—the least desirable, 10—the most desirable. In order to compare the sensory quality of the model meat products after 3 weeks of cold storage the following was applied: two flavor discriminants (smoked, fatty, where 0—the least intense, 10—the most intense) and the following traits were assessed: flavor desirability, color tone, tenderness, taste desirability, general quality where 0—the least desirable, 10—the most desirable. The sensory assessment of the sausages after 3 weeks of cold storage was limited to seven discriminants due to the unacceptable taste and flavor of the control treatment of the product. Samples before assessment were stored in a room with 24 ± 1 °C for 30 min. Each time the assessing team obtained samples of the MSPM sausages, coded with a three-digit code, in the plastic 250 mL container with a lid, a dish with water for mouth rinsing and the assessment card. There were two slices of sausage; each slice was from a different sausage bar, in each container. The assessment was performed by a trained team of eight people and conducted in the sensory analysis studio with individual stands for assessors with artificial lighting. The sensory quality assessment of the model products was performed after production and after 3 weeks of storage.
2.11. Statistical Analysis
All the experiments were performed in triplicates in the independent trials (replicates) n = 3 and a completely randomized design were used. A one-way analysis of variance (ANOVA) was applied, first between treatments and then between the storage time of the samples for all parameters. Means and standard deviations were calculated and a probability level of p < 0.05 was used in testing the statistical significance of all of the experimental data. The Fisher test was used to determine the significance of the mean values for a multiple comparison (p < 0.05). Data analyses were conducted using the STATGRAPHICS v. 4.1 statistical program (Manugistics Inc., Rockville, MD, USA).