Nisin In situ activity and quantification
Agar diffusion method is the most widely used method to determine nisin activity as well as for the quantification of nisin, due its simplicity and cost-effectiveness. This method is based on the measurement of the inhibition zone produced in nisin sensitive strain entrapped 49 in poured Petri dishes. The size of this zone is affected by many factors, such as the nisin sensitive strain used, the amount of agar and the pre-diffusion step. Lactobacillus sake was found to be the more nisin sensitive species compared with Micrococcus luteus and Brochothrix thermosphact (Pongtharangkul & Demirci, 2004).
However, poor accuracy of this assay limits the interpretation of results (Bouksaim et al., 1998). It is also time consuming and laborious, requiring preparation and cooling of plates, boring of test wells in agar and manual measuring of the inhibition zones after 24-48h of incubation (Tramer & Fowler, 1964). Results depend largely on human ability and judgment and the suggested precision cannot be obtained when the inhibition zone is unclear or not perfectly circular (Parente et al., 1995). Another method was proposed (Raheem & Saris, 2009) using the nisin producing strain Lactococcus lactis LAC309. The amount of nisin produced in Nigerian Wara cheese was measured by a nisin bioassay in which nisin induces the production of green fluorescent protein. Nisin-induced fluorescence was measured in cheese aqueous phase at 485 nm (excitation) and at 538 nm (emission) and was detected in terms of relative fluorescence units (RFU). The results in RFU were extrapolated to standard levels of Sigma commercial nisin stock solutions to obtain the amount of nisin in Wara cheese curd.
The nisin activity was quantified in many cheeses, mainly by the agar diffusion method. The results have been summarized in the Table (1).
Factors affecting nisin efficiency as a food preservative
Comparisons of data obtained in culture media with those obtained in food systems reveal that the efficacy of bacteriocins is often much lower in the later (Schillinger et al., 1996). Factors that may influence the recovery and efficiency of bacteriocins in foods are related to food components, and conditions that destabilize the biological activity, like proteolytic degradation or oxidation (Daeschel, 1993; Murray & Richard, 1997; Gänzle et al., 1999). Reduced activity of bacteriocins in foods was shown for nisin with high fat content (Bell & de Lacy, 1986; Jung et al., 1992; Davies et al., 1999). Nisin may also adsorb to proteins in the food matrix by ionic or hydrophobic bonds. These kinds of interactions and their effect on the inhibition efficiency have been less studied than the influence of fat, but Goff et al., (1996) and Murray & Richard (1997) demonstrated that protein binding may cause a significant reduction in free bacteriocin in foods. Addition of casein reduced the activity of nisin in synthetic media (Gänzle et al., 1999).
Nisin production and stability in a food matrix is also affected by several factors such as the producer lactococci strain, nutrient composition, pH, temperature, adsorption of nisin onto the producer cells or onto food component or enzymatic degradation (Parente et al., 1995; Schillinger et al., 1996). Among the factors influencing the effectiveness of bacteriocins as antimicrobials in food systems, factors influencing bacteriocin production are of most importance when using bacteriocinogenic cultures (Figure 3).
Homogenization of milk or cheese slurries was found to reduce nisin efficacy against Listeria monocytogenes comparing with non homogenized milk (Bhatti et al., 2004). It is known the homogenization decrease average diameter of fat globules and increase their numbers and surface area, which results in increment of the nisin adsorption on fat surface and hence decreased its overall antimicrobial activity. Some activity could be recovered by adding 0.2 v/v% of the Tween 80 emulsifier and to a lesser extent by adding lecithin (Glass & Johnson, 2004).
In agarose gel and food-like matrix
Nisin diffusivity was calculated in agarose gels only by taking into account the different factors capable to influence the diffusion, such as temperature, pH and agarose concentration in the gel (Sebti et al., 2004). Fick‟s second law was validated as a satisfactory mathematical model to assess nisin diffusion in such a model matrix (Carnet-Ripoche et al., 2006).
The experimental procedure used to determine the diffusivity of nisin is illustrated in Figure 4. The cylindrical geometry is usually chosen in order to be able to consider unidirectional mass transfers along the x-axis in the product. Before diffusion assay, the gels were coated with paraffin and parafilm layers in order to avoid evaporation of water.
Fat and NaCl contents
The influence of fat content in a model matrix (agarose) on nisin diffusivity was evaluated by Carnet-Ripoche et al (2006). The authors revealed that there was no significant difference between nisin apparent diffusion coefficients in 3% agarose gels with or without hydrogenated coprah oil (from 33 to 100% (w/w in the agarose) of hydrogenated coprah oil Vegetaline®) incorporated into 3% (w/w) agarose gel before gel preparation. D was 42 μm2/s n all treatments. For the authors, these results could be attributed to that the amount of nisin which was so high ( 277- 334 μg nisin/mL ) that apart of nisin saturated the fixation sites on Vegetaline®, the other part could diffuse easily. However, Chollet et al.,(2008) obtained different result when they examined the effect of fat and salt level on nisin recovery in cheese-like gels with 3% agarose and 2 different studied levels of anhydrous milk fat% and NaCl % w/w (5 and 30; 0.5 and 2, respectively) and Emmental cheese slurries. In their study, no significant differences were noticed according to the level of NaCl, while increasing anhydrous milk fat concentration in agarose gels caused nisin concentration to drop by a factor of 1.6, and also gave rise to a significant decrease in nisin bioactivity. The binding of nisin to fat seemed to limit its activity.
To prolong nisin efficacy, nisin has been incorporated into packaging films and coatings (Bi et al., 2011). Some results concerning nisin diffusion from the pachaging films into food matrixes were published.
In packaging films
Many food products can be subjected to contamination by undesirable microbes such as fungi, yeasts and bacteria (Hotchkiss, 1997). In order to prevent or impede such contamination, novel packaging technologies are continually being developed to prolong the shelf-life and improve the safety or sensory properties of fresh foods (Ahvenainen, 2003). A more recent and advanced class of food packaging system is known as “active packaging”(Hotchkiss, 1997).The active packaging has been defined as „„a type of packaging that changes the condition of the packaging to extend shelf-life or improve safety or sensory properties while maintaining the quality of the food‟‟(Quintavalla & Vicini, 2002).
To determine nisin diffusion in these active packing, a thin film was immersed in water. Nisin concentration into water over time is estimated. The mass transfer was modeled thanks to the Fick‟s second law. Many similar aspects were found between nisin diffusion in active packaging and in agarose gels. Temperature was found to have the same effect on nisin diffusion in both packaging films and agarose gels. Teerakam et al., (2002) showed that nisin diffusion coefficients ( D ) were increased when temperature was elevated. Diffusion was calculated in different protein films [cost corn zein (CCZ), heat-pressed corn zein (HPCZ), cast wheat gluten (CWG), and heat-pressed wheat gluten (HPWG)] at several temperatures from 5 to 45°C. D values ranged from 5.6 to 1123 x 10-5μm2/s. The same trend was also obtained by Dawson at al., (2003). It can be noticed that the rate of diffusion is much lower in polymeric films, and even more in proteinic ones, than in model food systems like agarose gels.
Also, an inverse relationship was found between nisin diffusion and the concentration of its constituent in active packaging as well as in agarose gels. Buonocore et al., (2003) investigated the release kinetics of antimicrobial agents (including nisin) from crosslinked polyvinylalcohol (PVOH) film into water at ambient temperature (25°C) and under moderate stirring. The tested films had different % of PVOH 0.077; 0.77, 2.00 and 7.70 % w/w. The obtained D values were 86.10; 62.40; 31.60 and 3.01 x 10-3 μm2/s, respectively.
Dry matter content and pH
Four cylindrical blocks of each type of cheese (R and R-G) were prepared as described in experimental device for nisin diffusion section. The moisture content was measured in duplicate in 1.5 ± 0.2 g slices of the R and R-G cheeses after 3 and 6 days incubation with the nisin solution (19). Both model cheeses had the same final dry matter (21.0 ± 0.5%) and the same pH, which was measured using a pH meter (inoLab pH Level 1, WTW®, Germany) with an accuracy of ± 0.01. Nisin concentration was recalculated for the aqueous phase of the cheese matrix, taking the dry matter content of each slice into account. The dry matter content in each slice was uniform throughout the cheese cylinder prior to the diffusion process. However, it increased in the slices close to the exposed surface after a few days of incubation due to a water intake from the nisin solution into the product (data not shown).
Nisin extraction from model cheese slices
Beginning with the exposed surface in contact with the nisin solution, the cheese cylinders were cut into thin slices. Each slice was weighed (1.5 ± 0.2 g) and its thickness precisely measured using a caliper rule (about 2 mm thick). The slices were then grated and diluted 1:10 (w/w) in acidified citrated water (pH=5) before homogenization with an Ultra-Turrax T25 (IKA Labortechnik, Staufen, Germany) for 2 min at 8500 rpm. The extract was centrifuged at 6000 g for 10 min. For each sample, 1 mL aliquots of the supernatant were stored for several weeks at -80°C until analysis.
Table of contents :
1. Diffusion of solutes in cheese:
1.1. State of the art: Determination of the diffusion coefficients of small solutes in cheese: A review (article)
1.2. Solutes diffusion and cheese macro-and microstructure
2. Nisin: a biological tool for food bio- preservation
2.1. Nisin, a bacteriocin of lactic acid bacteria
2.2. Nisin chemical properties
2.3. Nisin mode of action
2.4. Nisin applications
2.5. Nisin in situ activity and quantification
2.6. Factors affecting nisin efficiency as a food preservative
3. Diffusion of nisin in a solid matrix
3.1. In agarose gel and food-like matrix
Different factors may affect nisin diffusion
ii. Agarose content%
iii. Fat and NaCl contents
3.2. In packaging films
Chapter 1: Nisin diffusion in UF model cheese
Nisin quantification by ELISA allows the modeling of its apparent diffusion coefficient in model cheeses (article)
Chapter 2: Nisin efficacy in UF model cheese
The concentration of active nisin does not allow the prediction of its in situ efficiency in model cheese (article)
General conclusion and perspectives