Bio-preservation potential of Lactococcus lactis FO1 bacteriocin in fish sausage with optimizing production condition

Abstract

In response to growing interest in bio-conservation as a means of limiting the use of chemical preservatives, this study optimized the formulation of a fish sausage using a bacteriocin derived from Lactococcus lactis F01. The optimized “BIO Opti” formulation was then compared to a non-optimized organic sausage, a formulation containing sodium benzoate “CHE”, and a control. The optimization, conducted using a centered composite design, determined that the optimal conditions are 327.015 µg of bacteriocin per 50 g of product and 5.02% NaCl. Under these conditions, “BIO Opti” exhibits a significantly lower microbial load of 1.56 ± 0.84 log10 CFU/g and a moisture content of 61.55 ± 0.09%, compared with 1.75 ± 0.72 log₁₀ CFU/g and 62.55 ± 0.31% for BIO (p< 0.05). Furthermore, after 28 days of storage at 8 °C, the sensory quality of “BIO Opti” remains equivalent to that of “CHE” (p > 0.05). Taken together, the results indicate that optimization extends shelf life and positions bacteriocin as a credible natural alternative to synthetic preservatives.

1 Introduction

Fish and fishery products contribute to about 60% of the world’s animal protein supply. Thus, one of the most consumed fishery products today is fish, thanks to its high content of omega-3 essential fatty acids, its easily digestible proteins, and its content of essential amino acids (Adedeji et al., 2011). It is also rich in minerals (iron, phosphorus, zinc, copper), trace elements (fluorine, selenium), and vitamins (Rieu, 2012). Despite these nutritional values, fish is a highly perishable fishery product after their capture (Prabhakar et al., 2020). Indeed, external (storage conditions, temperature, and storage materials) and internal factors (protease, autolysis lipase, pH, and spoilage or pathogenic flora) can be the cause of this spoilage (Goulas and Kontominas, 2007). As a result, households and the agri-food industry have adopted many processing techniques to minimize post-capture losses by diversifying fish products (sausages, meats, pâtés, etc.), but also to increase their shelf life. For this reason, the food industries use chemical preservatives (sodium nitrate, sodium benzoate, etc….) to make these products permanent on the markets. However, these have adverse effects on the health of consumers (eye irritation, asthma, digestive disorders, behavioral disorders, insomnia, hyperactivity in children, and some cancers (colorectal cancer) (EFSA ANS panel (EFSA panel on Food additives and Nutrient Sources added to Food), 2013) (Yunsuke et al., 2019);. However, the current consumer trend to prefer food products with less or no chemical preservatives, coupled with the ban on many synthetic (chemical) preservatives by European legislation [European Union Directive (EC) No. 1107/2009], has driven the search for more natural means of preservation (European Parliament and the Council of European Union, 2009). It is in this perspective that studies have focused on the ability of strains of lactic acid bacteria and their peptide metabolite (bacteriocin which are extracellular antimicrobial peptides synthesized at the ribosomal level) to limit the growth of certain spoilage germs and/or pathogens in certain fishery products. Indeed, some works have highlighted the preservative power of BAL strains such as the work of (Nga et al., 2018) which focused on tropical pink shrimp in Cameroon; or the work of (Noraphat et al., 2015) which showed the ability of antibacterial peptides to inhibit the growth of spoilage or pathogenic bacteria in cooked peeled shrimp. Similarly, the work of (Diop et al., 2015; Diop et al., 2016) showed the effectiveness of the combination of NaCl and bacteriocin to inhibit the growth of pathogens or spoilage agents on fresh fish fillets in Senegal. To the same end, the work by (Kaktcham et al., 2019) focused on the biopreservation of fish “pâté” using a bacteriocin as a bio preservative. Despite the importance of these natural preservatives, products undergoing biopreservation often have a short optimal shelf life (OSL). This short duration is illustrated by the work of (Raju et al., 2003) on fish sausage (Ekhtiarzadeh et al., 2012), on fish fillets, and (Kaktcham et al., 2019) on “fish pate.” Therefore, the following question was asked: can optimization of production conditions extend the optimal shelf life of these foods? Thus, our objective was to optimize the production conditions of fish sausage using the concentration of bacteriocin produced from Lactococcus lactis FO1 and NaCl as a factor.

2 Materials and methods

2.1 Strain culture and growth

The bacteriocin-producing strain Lactococcus lactis FO1 and the indicator strain Lentilobactacillus senioris were isolated from the gastrointestinal tract of carp (Cyprinus carpio). The fish sample was collected from fish farmers in the town of Bâtie 5° 18’ 53’’ North, 10° 19’ 31’’East (West Cameroon) and then cultured on MRS broth and incubated for 24 h at 37 °C. Both strains were kept in MRS agar plates (Hi-Media) and propagated as needed.

2.2 Preparation of bacteriocin and determination of antibacterial activity

Two milliliters of Lactococcus lactis F01 culture were transferred into 500 ml of MRS broth. The culture broth was centrifuged after growth (14 h at 37 °C; pH 6.5) at 10000 rpm for 15 min and the cell-free supernatant was collected. The supernatant was adjusted to pH 7.0 with NaOH (0.1 N) and was sterilized by filtration (0.45 µm pore size) under vacuum. The bacteriocin was partially purified by double precipitation, first with ammonium sulfate and second with Chloroform-methanol and then the antibacterial activity was determined. To detect the activity of the bacteriocin, the methods described by Kaktcham et al. (2012) and Grosu-Tudor et al. (2014). were used. Briefly, 10 µl of the cell-free filtrate was spotted on an agar plate containing the susceptible strain. The inhibitory activity against indicator organisms was observed after incubation of the agar for 24 h at 37 °C and was expressed in arbitrary units (AU/;ml) this part has been performed following the method described by Fotso et al. (2022).

2.3 Optimization of production condition of fish sausage using bacteriocin produces from Lactococcus lactis FO1

2.3.1 Choice of factors and experimental domain

Based on the previous studies and literature data (Diop et al., 2015; Diop et al., 2016), NaCl level and bacteriocin concentration were selected as independent variables. A central composite design (CCD) was then applied to determine the optimal condition extending the shelf life of fish sausage. The responses evaluated were bacterial growth, pH, and moisture content. The ranges of the different independent variables used are presented in Table 1. A total of 13 experimental runs with five replications at the central point (Table 2) were performed. The results were the average of triplicates.

2.3.2 Preparation of fish sausage samples

Sausages were prepared using the different ingredients. The ingredients were added at the following final concentration: wheat flour is equal to 12.94% of (flesh, skin, oil, brine); brine is equal to 450 g/kg of flesh; pepper is equal to 10 g/kg of flesh; garlic is equal to 6 g/kg of flesh; oil is equal to 25 ml/kg and finally; the salt is equal to 3.2% of (flesh, skin, oil, brine). The preservative (bacteriocin) was added at 1.5 ml/50 g and the cooking was carried out at a temperature of 80 °C for 45 min.

3 Evaluation of responses

3.1 Evaluation of the bacterial load

ISO standards were used to determine the microbiological load of the following bacteria during 28 days: aerobic mesophyl flora total (FMAT) by the ISO Standard (NF ISO 4833), Salmonella sp. (NF ISO 21567), Staphylococcus sp. (NF V-08-057-1), Vibrio sp. (NF ISO 21872-1), and Escherichia sp. (NF V 08053). A 25 g slice of the preserved fish sausage was ground under aseptic conditions in a mortar with a sterile pestle and homogenized for 2 min with a vortex. Samples were serially diluted and plated in triplicate on nutrient agar (Plate count Agar Hi-Media) for FMAT; the Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar (Hi-Media) for Vibrio sp., Salmonella-shigella agar (SSA Hi-Media) for Salmonella sp, Mannitol Salt Agar (MSA Hi-Media) for Staphylococcus aureus and Mann Rogosa Sharpe (MRS; Hi-Media) for Lactococcus lactis sp. Cultures were incubated at 37 °C for 48 h for FMAT and 24 h for the other bacterial microorganisms being tested.

The total number of colonies present in the sample unit is given by the formula below:

Where

n1: number of plates counted at the lowest retained dilution, n2: number of plates counted at the second retained dilution, d: dilution rate at which the first counts are made: lowest dilution, ∑C: total number of colonies on the retained plates, V: volume of the inoculated test sample in ml, VSM: volume of the stock suspension in ml, VPR: mass of the product (g) that made up the stock suspension.

3.2 Determination of pH and moisture content

The different samples stored at 8 °C were analyzed for changes in pH and water content during the 28-day storage period. 1 g of each sample was ground and then introduced into 9 ml of distilled water and the mixture was homogenized by vortexing. The pH was determined using a pH meter (HI 2211 pH / ORP meter, HANNA instruments). For the water content, 5g of each sample was deposited in hollows and then deposited in a ventilated oven (Binder ED-S) so the temperature was 105 °C and this until a stable mass was obtained.

4 Postulated model and validation

The data were fitted to a second degree polynomial model (Y) as shown below:

Where Y represents the response (bacterial load, pH and Moisture content); β0 is the constant; β1 and β2 are the linear coefficients; β11 and β22 are squared coefficients; β12 the interaction coefficient; X1, X2, X₁², X₂² and X1.X2 are the levels of independent variables. To check the validity and robustness of the predictive model, the coefficient of determination (R2), bias factor (Bf), and mean absolute deviation (MAD) were used. Concerning the validation of the optimal conditions, three experimental replicates were performed under optimized condition and the level of significance of the obtained value was compared to the predicted results proposed by the model.

4.1 Comparative shelf-life study of the optimized fish sausage, non-optimized fish sausage (partially purified bacteriocin-NaCl-sausage), sodium-benzoate fish sausage and control fish sausage

The mixture was divided into batches of 250g each in artificial casings before cooking:

Batch distribution

• - Batch 1 control consisted only of sausage without preservative

• - Batch 2 consisted of non-optimized fish sausage + partially purified bacteriocin + Nacl (Bio) [1.5 mL/50 g = 253.5 µg/50 g and 4% NaCl].

• - Batch 3 consisted of optimized fish sausage + partially purified bacteriocin + Nacl (Bio Opti) [1.935 mL/50 g = 327,015 µg /; 50 g and 5.02% NaCl].

• - Batch 4 was fish sausage + sodium benzoate (CHE) [1 g/kg] (The Japan Food Chemical Research Foundation, 2018).

These different batches were then cooked at 80 °C for 45 min and finally stored at 8 °C for 4 weeks, during this time, testing was performed after every 7 days. The following parameter were evaluated the pH, the water conten, bacterial load (total mesophilic aerobic flora, Salmonella sp. Staphylococcus sp., Vibrio sp. and Escherichia sp.) and sensory parameters. It should be highlighted that, the evaluated of bacterial load was performed as previously described in Sections 2-5–1 and 2-5-2.

For the sensory evaluation, quantitative descriptive test made up of 15 trained people was used. Their recruitment was based on criteria such as interest and motivation, availability, intellectual ability, health status, and absence of anomalies or alterations affecting the senses. In addition, they were trained to assess the intensity of each of the descriptors, to be able to determine small differences in intensity and to give identical responses for the same product (repeatability). Using a five-point rating scale ranging from “very poor” to “very good,” the panel evaluated the color, texture, and odor of the products.

4.2 Statistical analysis

The variations between different samples during storage were verified by statistical analysis. Means and standard deviations were calculated using Microsoft Office Excel 2013. Minitab 18 and Sigmat plot software was used for the experimental design; then the power of the model was evaluated by assessing the coefficient of determination (R²) obtained from the analysis of variance (ANOVA). Using the same software, analysis of variance test (ANOVA) was done on the result to determine influencing factors on the responses. This was followed by comparisons of the means between the predicted and experimental value using IBM SPSS Statistics 22 software at 0.05 probability thresholds.

5 Results and discussion

5.1 Preparation of bacteriocin and determination of antibacterial activity

(Table 3) shows the results of the partial purification (amount of protein, yield, specific activity, purification factor, and total activity) following the different purification steps. The production of bacteriocin by Lactococcus lactis FO1 in MRS broth was maximal after 12 h of incubation. A gradual stationary phase and a steady decline phase were obtained after 14 h. An increase in protein concentration from 25.7 µg/ml in the cell free supernatant (CFS) to 86.8 µg/ml in the ammonium sulfate precipitate (ASP) and from the latter to 169 µg/ml in the chloroform-methanol precipitation (CMP) was observed. This increase is also observed in the specific activity, which is 31.12 UA/µg in CFS to 37.82 UA/µg in PSA and from the latter to 37.86 UA/µg in CMP with a purification yield of 40%. These results can be explained by the reduction of impurities after precipitation, resulting in an increase in activity. Furthermore, its antibacterial activity can be explained by its nature; given its nature and class (I), it binds to the membrane of pathogenic or harmful bacteria via the disaccharide-pyrophosphate of lipid II and disrupts the transport or synthesis of peptidoglycan, which is a very important component of the membrane. On the other hand, by creating pores in the membrane, it disrupts the acid-base balance in the cytoplasm and directly causes cell death (Grosu-Tudor et al., 2014; Kaktcham et al., 2019; Jinjin et al., 2021; Gutiérrez-Cortés et al., 2018) obtained observations along these lines.

5.2 Optimization of production conditions of fish sausage using bacteriocin produced from Lactococcus lactis FO1

5.2.1 Experimental values of the response measured during the experimental tests

The bacterial load obtained after the 13 experimental trials is summarized in Table 4. It appears that the bacterial load ranged from (2.37± 0.90 and 1.61 ± 0.62) log₁₀ CFU/g for FMAT, from (1.90 ± 0.79 and 0) log₁₀ CFU/g for Staphylococcus sp. and from (2.16 ± 0.98 and 0) log₁₀ CFU/g for Vibrio sp. Furthermore, this table also shows that the lowest bacterial load was obtained when the volume of bacteriocin was 2 ml and the NaCl concentration was 5%. However, the pH and water content varied very little for all samples. The analysis of the obtained results allowed us to determine the influence of the different factors and the conditions required to obtain a low bacterial load, pH and water content.

These results have as explanations the bactericidal effect of bacteriocin works such as Ekhtiarzadeh et al. (2012), Grosu-Tudor et al. (2014), and Kaktcham et al. (2019). In addition, NaCl potentiates the action of bacteriocin, as it is observed that increasing the concentration of NaCl and bacteriocin up to a certain value significantly reduces the bacterial load and allows for a better pH and water content. A similar observation was obtained by Diop et al. (2015) and Diop et al. (2016) on the effectiveness of the combination of NaCl and neutralized culture supernatants of lactic acid bacteria to inhibit the growth of pathogens or spoilage agents on fresh fish.

5.2.2 Proposal of the mathematical model and contribution of the different factors on the variation of the response

The mathematical equation resulting from the optimization and which can predict the variation of the bacterial load, pH and Moisture content is presented below.

With Y: bacterial load; A: the volume in bacteriocin; B: the concentration in NaCl.

5.2.3 Pareto analysis and model validation

The analysis of variance allows us to highlight the effect of the different control variables. In Figure 1e, we can observe that the FMAT is influenced by the linear effect of NaCl, and the linear effect of bacteriocin influences the load of Vibrio sp. and S.A. The quadratic effect of bacteriocin influences the FMAT while the quadratic effect of NaCl impacts the load of S.A and Vibrio sp., the interaction between bacteriocin and NaCl has an effect on the load of FMAT and S.A. However, the linear and quadratic effects of bacteriocin do not influence pH and water content, but the linear effect of NaCl influences pH and water content. Indeed, bacteriocin is the factor with the most effect on one of the responses because it has a major impact on the bacterial load. The validation of the model is one of the fundamental steps of the experimental design. It consists in comparing the theoretical result of an experiment calculated by the model with the real result of a test. The values of the different elements of the model validation are grouped in Table 5. From this table, it can be seen that for the model, the coefficient of determination (R²) for the optimization of the bacterial load was 89.51(FMAT), 92.57(S.A), 83.67(Vibrio sp.), 83.27(pH) and 95.57(water content). In addition, the absolute analysis of the mean deviation gave the value of 0.00 for all the responses. Regarding the bias factor, the value obtained was 0.97(FMAT); 1 (S.A); 0.93 (Vibrio sp.); 0.92(pH) and 1 (water content) (Joglekar and May, 1987).

Figure 1

5.2.4 Effect of the different factors on the bacterial load,

Figure 2c shows the effects of bacteriocin and NaCl on FMAT, Vibrio sp., and S.A load respectively. It can be observed that the bacterial load (FMAT, Vibrio sp., and S.A) decreases with the increase of the amount of bacteriocin up to the value of 1.6 ml; however, this bacterial load increases beyond this value for FMAT and Vibrio sp. but continues to reduce the load of S. A. The results presented in the figures are explained by the bactericidal effect of bacteriocin on bacteria because it exerts its bactericidal action through several mechanisms among which the perforation of the membrane of the bacteria thus disturbing the acid-base balance in the cytoplasm of the bacteria or they can also bind to certain molecules and stop the synthesis of certain molecules essential to the bacterial cell (Sarika et al., 2019).

Figure 2

5.2.5 Effect of the different factors pH and moisture content

The NaCl reduces the load of these bacteria, pH, and water content. From these Figures 2d, e, it is clear that increasing the NaCl concentration reduces the bacterial load and significantly lowers the pH and water content, while increasing the amount of bacteriocin does not affect pH and water content. Regarding the effect of NaCl, it has a double action: the first one can reduce the water content and consequently the water activity which is an essential factor for the bacterial growth, and the second one by modification of the pH of the medium which can favor or not the growth of certain bacteria. Work in the same direction has been reported by Diop et al. (2016).

5.2.6 Optimum conditions and validation of optimum conditions

The Figure 3 below represent the iso-response curves for bacterial load, pH and water content according to the interactions influencing them. The maximum activity is materialized on the figure by the hatch area with lines of different colors. This area of interest corresponds to the portion of the experimental area where the application of the relevant conditions will contribute to obtain a low bacterial load, a good pH and a better water content. From this figure, it can be seen that in order to obtain a sausage with good bacterial quality, acceptable pH, and good water content, the following optimal conditions are required: a NaCl concentration of 5.02% and a bacteriocin concentration of 169 µg/;ml, or 327.015 µg /; 50 g. These results are explained by the inhibitory effect of bacteriocin which inhibits the growth of bacteria and NaCl which has the ability to absorb water molecules reducing the amount of free water as well as ions in the medium and consequently varies the pH of the medium which positively or negatively promotes the growth of microorganisms (Diop et al., 2016).

Figure 3

5.2.7 Validation test of the optimal conditions

To confirm the agreement between theory and experiment, additional laboratory manipulations were performed following the optimal conditions predicted by the mathematical model (Table 6). The obtained optimal experimental values were compared with the predicted values to validate the optimal conditions defined by the model and it was found that no significant difference was observed between these values (p >0.05).

5.3 Comparative shelf-life study of the optimized sausage, non-optimized sausage (purified bacteriocin-NaCl sausage), sodium-benzoate sausage and control sausage

5.3.1 Microbiological analysis

A load of total aerobic mesophilic flora (FMAT), Staphylococcus sp., Vibrio sp., Salmonella sp., and Escherichia sp. was monitored in the different samples stored at 8 °C for 28 days and the corresponding results are presented in Figures 4a–c and summarized in Table 7. It can be seen that from day zero to day 28, the FMAT load varied from (2.71 ± 1.0 to 1.56 ± 0.84) log₁₀CFU/g for the optimized sample. From (2.71 ± 1.00 to 1.75 ± 0.72) log₁₀CFU/g for the non-optimized sample (both of these have bacteriocin as preservative) and from (2.71 ± 1.0 to 1.93 ± 0.73) log₁₀CFU/g for the sample having sodium benzoate as preservative. Similarly, the load of the control (sausage without preservative) increases from (2.71 ± 1.0 to 4.96 ± 1.20) log₁₀CFU/g over the 28 days. However, the optimized sample and the non-optimized sample do not differ significantly (p>0.05) from each other and also from the sample that received sodium benzoate as a preservative. But the samples that received preservatives (bacteriocin and sodium benzoate) differed significantly (p<0.05) from the control.

Figure 4

Regarding the Staphylococcus sp. load of the different samples, it ranged from (2.12 ± 0.90 to 0) log₁₀CFU/g for the optimized sample, from (2.12 ± 0.90 to 1.56 ± 0.30) log₁₀CFU/g for the non-optimized sample, and from (2.12 ± 0.90 to 1.65 ± 0.61) log₁₀CFU/g for the sausage with sodium benzoate as a preservative. On the other hand, in the sample without preservatives, the load evolved from (2.12 ± 0.90 to 3.22 ± 0.69) Log₁₀CFU/g. Indeed, it can be seen that the samples with bacteriocin as a preservative preserved the fish sausages better, but the optimized sample differed significantly (p<0.05) from the non-optimized sample, the sample with sodium benzoate as a preservative, and even the control.

Analyses were also performed to assess the Vibrio sp. load, which was found to vary from (1.91 ± 0.18 to 0) log₁₀ CFU/g for the optimized sample, from (1.91 ± 0.18 to 1.11 ± 0.21) log₁₀ CFU/g for the non-optimized sample, and from (1.91 ± 0.18 to 1.21± 0.10) Log₁₀ CFU/g for the sample with Sodium Benzoate as a preservative. For the sample without preservatives, the loading fluctuated between (1.91± 0.18 and 3.26 ± 0.71) log₁₀ CFU/g during the 28 days of storage at 8 °C. However, it is observed that the Salmonella sp. and Escherichia sp. load was zero from day zero. Furthermore, it was observed that the optimized sample differed significantly (p<0.05) from the non-optimized sample as well as from the sample with sodium benzoate as the preservative. However, the samples that received preservatives (bacteriocin and sodium benzoate) differed significantly (p<0.05) from the control.

In fact, these results show that it exerts its bactericidal effect through several mechanisms, including perforation of the bacterial membrane, thereby disrupting the acid-base balance in the bacterial cytoplasm. It can also bind to certain molecules and halt the synthesis of certain molecules essential to the bacterial cell. As for the effect of NaCl, it has a dual action: first, it can reduce water activity and consequently reduce the population of microorganisms, since water, activity is an essential factor for their growth—it determines the minimum amount of water available for their growth. Similarly, water influences chemical and enzymatic reactions in the environment because, in reactions, it can act as a solvent or reactant, or alter the mobility of reactants in solution, thereby affecting viscosity and the action of certain enzymes. The second effect involves changing the pH of the environment, which may or may not promote the growth of certain bacteria. However, the bactericidal effect of bacteriocin is the most important because the sausages with bacteriocin as a preservative have a low load compared to those with sodium benzoate as a preservative. The change in bacterial load after day 21 in the case of FMAT can also be explained by the increase in moisture content and pH, which can impact the activity of bacteriocin and even sodium benzoate. In addition, other factors intrinsic to the food may inhibit or reduce the activity of bacteriocin or sodium benzoate as they interact with certain food additives or ingredients. In general, it is observed that the load of the various pathogenic bacteria evaluated is lower than the standard during the 28 days of storage at 8 °C as is the case of Vibrio sp. whose load obtained was significantly lower (p<0.05) than that recommended for Vibrio Parahaemolyticus in the U.S. (100–000 CFU/g). Work along these lines has been reported by XI (2011), Nga et al. (2018), Kaktcham et al. (2019), and Sarika et al. 2019.

5.3.2 Evolution of moisture content (MC) and pH

The Moisture content (MC) of the different fish sausage samples stored at 8 °C for 28 days is presented (Figure 5a). It can be seen that the MC varied from 63.65 ± 0.81% to 61.55 ± 0.09% for the optimized sample, from 63.65 ± 0.81to 62.55 ± 0.31% for the non-optimized sample, and from 63.65 ± 0.81% to 66.46 ± 0.09% for the one containing sodium benzoate as preservative. However, in the sample without preservatives, the moisture content varied between 63.65 ± 0.81% and 66.30 ± 0.15% during the storage period. Thus, it can be seen that the moisture content of the optimized sample differed significantly (p<0.05) from the non-optimized sample, the sample with sodium benzoate as a preservative and the control, but the samples with bacteriocin as a preservative differed significantly (p<0.05) from the sample with sodium benzoate as a preservative and the control.

Figure 5

However, Figure 5b shows the pH variations in the different samples; it can be seen that the pH varied from 6.80 ± 0.01 to 6.82 ± 0.02 for the optimized sample, from 6.80 ± 0.01 to 6.95 ± 0.01 for the non-optimized sample, and from 6.80 ± 0.01 to 7.03 ± 0.03 for the sample with sodium benzoate as preservative. For the control sample, the pH varied from 6.80 ± 0.01 to 7.05 ± 0.03 during storage. However, it is observed that the pH of the samples with bacteriocin as a preservative are not significantly different (p > 0.05) from each other, but differed significantly (p<0.05) from the sample with sodium benzoate as a preservative and the control. This small variation in moisture content and pH of the samples is related to the input or composition of the raw material (fish) because the latter is low in carbohydrates that during catabolism lead to the production of acid, hence the variation in pH. Similarly, the composition of the elements used in the manufacture of fish sausage (spices, flour, egg, oil…) is also the cause of this small variation in moisture content and pH. Similar work has been reported by Nowsad et al. (2000), Gutiérrez et al. (2007), and Sarika et al. (2019).

5.4 Sensory evaluation

The color, odor, texture and general appreciation of our different samples, are reported in Figure 6d. The sausages treated with sodium benzoate and those treated with bacteriocin were able to maintain their good characteristics after 28 days of storage at 8 °C; but the sausages treated with bacteriocin were the best. However, we found that there was no significant difference (p>0.05) between the different treated samples with respect to the parameters evaluating color, texture and smell. These results are in agreement with those of Rodgers, (2001), Vermeiren et al. (2004), and Sarika et al. (2019) who in their work report that to use a bacteriocin as an additive; it should not change the color, flavor or odor of food. The decrease of the parameters evaluated in the present work during the conservation could be due to the microbial flora that during the metabolism produces substances, which can be responsible for the modification of the organoleptic characteristics in particular Likewise, the increase in water content, pH, relative humidity and oxidation by enzymes (enzymatic browning, lysis, destruction of vitamins and some nutrients) would also be responsible for changes in texture, smell and smell of the product. Work along these lines has been reported by Noraphat et al. (2015).

Figure 6

6 Conclusion

At the conclusion of this study, the optimization of the formulation parameters specifically, 327.015 µg/50 g of bacteriocin and 5.02% NaCl led to the development of a poison sausage (BIO Opti) with superior performance. Compared to the BIO and CHE formulations and the control, (BIO Opti) exhibited the best microbiological control with 1.56 ± 0.84 (FMAT) log₁₀ CFU/g and an optimal moisture content of 61.23 ± 0.06, with a significant difference (p< 0.05). Furthermore, the organoleptic stability over the 28 days at 8 °C, comparable to that obtained with sodium benzoate (p > 0.05), confirms the effectiveness of the process. These results demonstrate that the optimization extends the optimal shelf life and validate the bacteriocin produced by Lactococcus lactis F01 as a natural, effective, and safe alternative to chemical preservatives in fish sausages.

References

• 1

  AdedejiO. B.AdebisiT.EmikpeB. O. (2011). Bacteria load on the skin and stomach of Clarias Gariepinus and Oreocrhromis Niloticus from Ibadan, South West Nigeria: Public health applications. Jounal Microbiol. Biotechnol. Res.1, 52–59.

  • Google Scholar

• 2

  DiopM. B.AlvarezV. B.GuiroA. T.ThonartP. (2016). Efficiency of neutralised antibacterial culture supernatant from bacteriocinogenic lactic acid bacteria supplemented with salt in control of microorganisms present in Senegalese artisanally handled fish by immersion preservative technology during Guedj seafood processing at 10 °C and 30 °C. J. Food. Microbiol.1, 1–10. doi: 10.4172/2476-2059.1000102

  • CrossRef
  • Google Scholar
  • View reference in article

• 3

  DiopM. B.DestainJ.AlvarezV. B.KonteM. A.ThonartP. (2015). Use of nisin-producing starter cultures of Lactococcus lactis subsp. lactis on cereal based-matrix to optimise preservative factors over fish fermentation at 30 °C typical to Senegal. J. Food. Process. Technol.6, 432. doi: 10.4172/2157-7110.1000432

  • CrossRef
  • Google Scholar
  • View reference in article

• 4

  EFSA ANS panel (EFSA panel on Food additives and Nutrient Sources added to Food) (2013). Scientific Opinion on there-evaluation of aspartame as a food additive. EFSA J.11, 34–96, 263.

  • Google Scholar
  • View reference in article

• 5

  EkhtiarzadehH.BastiA. A.MisaghiA.SariA.KhanjariA.RokniN.et al. (2012). Growth response of Vibrio parahaemolyticus and listeria monocytogenes in salted fsh fllets as afected by Zataria multifora boiss. Essential oil, nisin, and their combination. J. Food Saf.32, 263–269. doi: 10.1111/j.1745-4565.2012.00376.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 6

  European Parliament and the Council of European Union (2009). “ Regulations: regulation (EC) no 1107/2009 of the European parliament and of the council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC,” in Of J Eur Union L, 1–50.

  • Google Scholar
  • View reference in article

• 7

  FotsoT. U. D.KaktchamP. M.MomoK. H.FokoK. E. M.TchamaniP. L.NgouenamJ. R.et al. (2022). Isolation, Characterization, and Effect on Biofilm Formation of Bacteriocin Produced by Lactococcus lactis F01 Isolated from Cyprinus carpio and Application for Biopreservation of Fish Sausage. BioMed. Res. Int.1, 8437926. doi: 10.1155/2022/8437926

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 8

  GoulasA. E.KontominasM. G. (2007). Effect of modified atmosphere packaging and vacuum packaging on the shelf-life of refrigerated chub mackerel (Scomber japonicus): biochemical and sensory attributes. Europ Food. Res. Technol.224, 545–553. doi: 10.1007/s00217-006-0316-y

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 9

  Grosu-TudorS. S.StancuM. M.PelinescuD.ZamfirM. (2014). Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods. World J. Microbiol. Biotechnol.30, 2459–2469. doi: 10.1007/s11274-014-1671-7

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 10

  GutiérrezA.NietoJ.PozoF.SternS.SchoofsL. (2007). Effect sofinsulin/IGF-I like peptides on glucose metabolism in the white shrimp Penaeus vannamei. Gen. Comp. Endocrinol.153, 170–175.

  • Google Scholar
  • View reference in article

• 11

  Gutiérrez-CortésC.SuarezH.BuitragoG.NeroA. L.TodorovD. S. (2018). Characterization of bacteriocins produced by strains of Pediococcus pentosaceus isolated from Minas cheese. Ann. Microbiol.68, 383–398.

  • Google Scholar
  • View reference in article

• 12

  JinjinP.wengangJ.JinzeW.YigangH.XinshengL.HongxiaZ.et al. (2021). Purification and characterization of plantaricin YKX and Assessment its inhibitory activity against Alicyclobacillus spp. Front. Microbiol.12, 783266. doi: 10.3389/fmicb.2021.783266

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 13

  JoglekarA.MayA. (1987). Product excellence through design of experiments. Cereal Food. World32, 857–868. doi: 10.1007/978-1-4613-0675-7_10

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 14

  KaktchamP. M.KouamE. M. F.TientcheuM. L. T.TemgouaJ. B.WacherC.NgoufackF. Z.et al. (2019). Nisin-producing Lactococcus lactis subsp. lactis 2MT isolated from freshwater Nile tilapia in Cameroon: Bacteriocin screening, characterization, and optimization in a low-cost medium. LWT107, 272–279. doi: 10.1016/j.lwt.2019.03.007

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 15

  KaktchamP. M.ZambouN. F.TchouanguepF. M.El-SodaM.ChoudharyM. I. (2012). Antimicrobial and safety properties of lactobacilli isolated from two Cameroonian traditional fermented foods. Sci. Pharm.80, 189–204. doi: 10.3797/scipharm.1107-12

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 16

  NgaO. S. N.KaktchamP. M.SeydiM.ZambouN. F. (2018). Changes in sensory, physicochemical, and microbiological properties of fresh captured tropical pink shrimps (Penaeusduorarumnotialis) inoculated with Lactobacillus plantarum Lp6SH, Lactobacillus rhamnosus Yoba, and their cell-free culture supernatants during storage at 4 °C. J. Food Saf.12, e12-579.

  • Google Scholar
  • View reference in article

• 17

  NoraphatH.JaffresE.DoussetX.PillotG.ChoisetY.HaertleT.et al. (2015). Application of a nisin Z-producing Lactococcus lactis subsp. lactis KT2W2L isolated from brackish water for biopreservation in cooked, peeled and ionized tropical shrimps during storage at 8 ^°C under modified atmosphere packaging. Europ Food. Res. Technol.240, 1259–1269. doi: 10.1007/s00217-015-2428-8

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 18

  NowsadA. A.HoqueM. E.SarkerF. C. (2000). First report of the formulation and development of fish sausage from underutilized marine fish in Bangladesh: fish sausage from sea catfish and Bombay duck. Bang. J. Fish.23, 107–112.

  • Google Scholar
  • View reference in article

• 19

  PrabhakarP. K.VatsaS.SrivastavP. P.PathakS. S. (2020). A comprehensive review on freshness of fish and assessment: Analytical methods and recent innovations. Food. Res. Inter.133, 109–157. doi: 10.1016/j.foodres.2020.109157

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 20

  RajuC. V.ShamasundarB. A.UdupaK. S. (2003). The use of nisin as a preservative in fish sausage stored at ambient (28±2 C) and refrigerated (6 ± 2 C) temperatures. InterJ Food. Scien Technol.38, 171–185. doi: 10.1046/j.1365-2621.2003.00663.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 21

  RieuD. (2012). Composition des poissons : protéines, lipides, vitamine D, iode…. Arch. Pediatry19, 36–37.

  • Google Scholar
  • View reference in article

• 22

  RodgersS. (2001). Preserving non-fermented refrigerated foods with microbial cultures: a review. Trends Food Sci. Technol.12, 276–284. doi: 10.1016/s0924-2244(01)00093-0

  • CrossRef
  • Google Scholar
  • View reference in article

• 23

  SarikaA. R.LiptonA. P.AishwaryaM. S. (2019). Biopreservative efficacy of bacteriocin GP1 of Lactobacillus rhamnosus GP1 on stored fish filets. Front. Nutr.6, 29. doi: 10.3389/fnut.2019.00029

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 24

  The Japan Food Chemical Research Foundation (2018). Standards for use of food additives24.

  • Google Scholar
  • View reference in article

• 25

  VermeirenL.DevlieghereF.DebevereJ. (2004). Evaluation of meat born lactic acid bacteria as protective cultures for the biopréservation of cooked meat products. Int. J. Food. Microbiol.96, 149–164. doi: 10.1016/j.ijfoodmicro.2004.03.016

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 26

  XID. (2011). Application of probiotics and green tea extract in post-harvest processes of Pacific oysters (Crassostreagigas) for reducing Vibrio parahaemolyticus and extending shelf life (USA: Oregon State University PhD thesis), 212.

  • Google Scholar
  • View reference in article

• 27

  YunsukeS.ShojiF.HijiriI.BarryL.AshleyR. (2019). Histological analyses of the Ishii (1981) rat carcinogenicity study of aspartame and comparison with the Ramazzini institute studies. Regul. Toxicol. Pharm.102, 23–28.

  • Google Scholar