# Study the effect of lactoperoxidase activation in combination with heat treatment or nisin on the microbiological quality of raw milk

Chedia Aouadhi, Souhir Kmiha, Faten Trakhna, Abderrazak Maaroufi

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Chedia Aouadhi, Souhir Kmiha, Faten Trakhna, Abderrazak Maaroufi. Study the effect of lactoperoxidase activation in combination with heat treatment or nisin on the microbiological quality of raw milk. Applied Food Research, 2024, 4 (2), pp.100521. 10.1016/j.afres.2024.100521. pasteur-05008878

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# Study the effect of lactoperoxidase activation in combination with heat treatment or nisin on the microbiological quality of raw milk

Chedia Aouadhi<sup>a,b,*</sup>, Souhir Kmiha<sup>a</sup>, Faten Trakhna<sup>a,c</sup>, Abderrazak Maaroufi<sup>a</sup>

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## ARTICLE INFO

### Keywords:

- Quality of milk

- Nisin

- Lactoperoxidase

- Thiocyanate

- Hydrogen peroxide

- Heat treatment

## ABSTRACT

The aim of the current study was to improve the microbiological quality of raw cow milk by activating the lactoperoxidase system using either nisin or heat treatment. A central composite experimental design with three factors was used to evaluate the effect of thiocyanate (SCN), hydrogen peroxide ($H_2O_2$) and nisin on the microbiological quality of raw milk. The experimental response was measured in terms of the logarithmic reduction in total microbial, total coliforms count, yeasts and molds. Our results indicate that the combination of both treatments was highly effective in destroying various microorganisms. In fact, the optimal values of each parameter (SCN, $H_2O_2$ and nisin) for a 6log, 5log and 4log reductions of the initial number of total aerobic mesophilic bacteria, coliforms, yeasts and molds were around 7 mg/L of SCN, 15 mg/L of $H_2O_2$ and 50 IU/mL of nisin, obtained after 72 h of incubation at 25 °C. Concerning the LPS activation at moderate temperature, our data suggest that this combination was very effective in destroying different microorganisms. Certainly, the total destruction of coliforms, yeasts and molds was noted after 96 h incubation at 25 °C after LPS activation for 4 h and 6 h followed by heat treatment (63 °C for 30 min). These results highlight a synergistic effect between the LPS activation and nisin or heat treatment in preserving microbiological quality of raw milk for 72 h or 96 h at 25 °C, respectively. Such treatments could be implemented by the dairy industry to enhance the quality of fresh milk and consequently of dairy products.

## 1. Introduction

Milk is a perishable product and an ideal medium for the growth of a wide variety of bacteria (Parekh & Subhash, 2008; De Silva et al., 2016). It is impossible to prevent the contamination of milk with microorganisms due to its specific production process; making its microbial content as a crucial determinant of its quality (Rogelj, 2003). Due to the lack of necessary facilities to maintain the milk fresh and therefore inhibit bacterial proliferation and sample deterioration prior to examination, these problems become very serious. In such situations, the use of antimicrobial agents such as the lactoperoxidase system (LPS) presents an attractive alternative conventional treatment for controlling microbiological quality of milk products in various countries (Aprodu et al., 2014; Lara-Aguilar & Alcaine, 2019; Fanta et al., 2019; Ahmed et al., 2021).

The antimicrobial activity of LPS naturally present in bovine milk and the saliva of humans and animals consists of lactoperoxidase (LPS), thiocyanate (SCN) and hydrogen peroxide ($H_2O_2$) (Köksal et al., 2017; Arefin et al., 2017). Its activation involves SCN⁻ oxidation by $H_2O_2$, which is catalysed by LPS. Hypothiocyanic acid (HOSCN) and hypothiocyanate ion (OSCN⁻): the compounds that have antimicrobial characteristics, are the products of this reaction (Seifu et al., 2005; Bafort et al., 2014; Al-Baarri et al., 2018).

This system significantly reduces bacterial load and extends the shelf life of raw milk at ambient temperature (Mohamed et al., 2013; Aprodu et al., 2014; Lara-Aguilar & Alcaine, 2019; Ahmed et al., 2021). In addition, its effectiveness depends on environmental conditions, target strains, temperature, LPO concentrations, generation mode of hydrogen peroxide and milk temperature during application (Aprodu et al., 2014; Lara-Aguilar & Alcaine, 2019; Ahmed et al., 2021). It possesses

<footnote><sup>a</sup> Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, University of Tunis El Manar (UTM), Pasteur Institute of Tunisia, BP 74, 13 place Pasteur, Belvédère, Tunis 1002, Tunisia</footnote>

<footnote><sup>b</sup> Institut Supérieur de Biotechnologie de Beja, Université de Jendouba, Beja 9000, Tunisia</footnote>

<footnote><sup>c</sup> Health Information Technology, Applied College, King Abdul-Aziz University, Jeddah, Saudi Arabia</footnote>

<footnote>* Corresponding author at: Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia, University of Tunis El Manar (UTM), BP 74, 13 place Pasteur, Belvédère, Tunis 1002, Tunisia.

E-mail address: chediaaouadhi@yahoo.fr (C. Aouadhi).</footnote>
significant bactericidal activity against Gram-negative bacteria such as E. coli and bacteriostatic efficacy against Gram-positive bacteria including L. monocytogenes and S. aureus (Seifu et al., 2004; Khudhir & Hammad, 2016; Ahmed et al., 2021), in dairy products, meat and synthetic media (Elliot et al., 2003; Seifu et al., 2004). As a matter of fact, to improve its antimicrobial effect, several researchers have explored this system in combination with other antimicrobial agents and thermal and non-thermal treatments. For example, activation of the lactoperoxidase system with nisin has been used to inhibit the growth of E. coli O157: H7 in milk (Khudhir & Hammad, 2016). Nisin, the only bacteriocin approved as a food preservative, has been recognized as GRAS (Generally Recognized As Safe) by the US Food and Drug Administration (FDA) since 1988 (Food & Drug Administration, 1998). It inhibits both the vegetative forms of pathogenic Gram-positive bacteria and the outgrowth of their spores (Aouadhi et al., 2015; Aouadhi & Maaroufi, 2023). Similarly, it shows effectiveness against Gram-negative bacteria when used in combination with chelators such as ethylenediaminetetraacetic acid (EDTA), heat treatment, freezing, LPS or any treatment causing the alteration of the cell wall. Consequently, the cell wall becomes permeable which promotes contact between nisin and the cytoplasmic membrane (Belfiore et al., 2007; Khudhir & Hammad, 2016).

To understand the effects of component concentrations utilized for the activation of LPS and nisin on raw milk quality, several approaches can be used. In microbiology, response surface methodology (RSM) appears to be particularly interesting. The application of RSM provides an empirical relationship between the response variable and the test variable under consideration (Aouadhi et al., 2013). Although this methodology has been employed to model the effects of lactoperoxidase system and nisin on the behavior of Listeria monocytogenes CIP 82110T by Boussouel et al. (1999) and to optimize the components concentrations of the lactoperoxidase system by Adolphe et al. (2006), evidently it has never been used to optimize the LPS activation and nisin against raw milk microflora.

The current investigation was conducted to study the LPS activation in combination with nisin or heat treatment (63 °C for 30 min) on the microbiological quality of raw milk incubated at 25 °C for 72 h or for 96 h, respectively. To do this, the response surface methodology was employed to determine optimal concentrations of these two components (SCN and H₂O₂) and nisin in order to improve of raw milk preservation.

## 2. Materials and methods

### 2.1. Milk sampling

The raw cow milk samples (39 samples), used in this study, were obtained from dairy plant “North Central Dairy” located in Bousalem, Jendouba (Tunisia) during peak lactation (March-April). Milk samples derived from a mixed raw milk provided from three collection centers. The physicochemical properties of the raw milk samples are summarized in Table 1.

### 2.2. Preparation of nisin solution

The stock solution of nisin (10⁴ IU/mL) was prepared by dissolving

<table><caption>**Table 1**
The physicochemical proprieties of raw milk samples.</caption><thead><tr><th>Parameter</th><th>Value</th></tr></thead><tbody><tr><td>pH</td><td>6.62-6.63</td></tr><tr><td>Acidity (°D)</td><td>14.5-15</td></tr><tr><td>Density</td><td>1.028</td></tr><tr><td>Antibiotic test</td><td>Negative</td></tr><tr><td>Fat (g/L)</td><td>30.25-33</td></tr><tr><td>Protein (g/L)</td><td>28.84-29</td></tr><tr><td>Lactose (g/L)</td><td>39.92</td></tr></tbody></table>

10 mg of nisin (Sigma, Aldrich) in 1 mL of sterile solution of HCL (0.02 N) (Aouadhi et al., 2015).

### 2.3. The activation of lactoperoxidase system

The activation of the LPS was realized by adding the SCN and H₂O₂ to raw milk in different proportions as indicated in Table 2 (Ahmed et al., 2021). The samples, following the addition of the SCN, H₂O₂ and nisin, were incubated at 25 °C for 72 h. Subsequently, samples were analyzed for microbiological parameters before and after treatment.

### 2.4. Microbial analysis

Microbial analysis was conducted according to standard methods as described by Aouadhi et al. (2024), Ahmed et al. (2021). Total viable counts in raw milk samples were estimated using plate count agar media (Biokar-Diagnostics), with plates incubated for 72 h at 37 °C. Coliform counts were determined by plate count method using violet red bile agar (Biokar-Diagnostics), prepared according to the manufacturer instructions, and all plates were incubated for 24 h at 37 °C. Sabouraud agar (Biokar-Diagnostics) was employed to enumerate of yeasts and molds with all plates being incubated at 25 °C for 5 days

### 2.5. Experimental design

A rotatable central composite design (CCD) for three factors (SCN concentration, H₂O₂ concentration and nisin concentration), as described by Aouadhi et al. (2013), was used to examine the effect of LPS activation and nisin on preserving the quality of raw milk. Each variable was tested at five coded levels (indicated as -1.68, -1, 0, +1, +1.68) as shown in Table 2 using the software STATGRAPHICS Centurion XV version 15.2.06 (Table 3) for experimental design and analysis. The real values of the independent variables (X) were coded according to Eq. (1):

$$x_i = (X_i - X_0)/\Delta X, \quad (1)$$

where Xi is the real value of the independent variable; xᵢ is the coded value of the independent variable given by the experimental matrix; X₀ is the real value of the central point, and ΔX is the step change (difference between central point (level 0) and level (-1) or level (1)). X and x values are shown in Table 2.

The experimental response was the logarithmic reduction in total bacterial count, coliforms count, yeasts and molds counts (log (N₀/N), with N₀ and N represent the number of each micro-organism before and after treatment, respectively). A full quadratic model containing 10 coefficients was utilized to describe observed responses and to fit the Eq. (2):

$$Y = b_0 + b_1 \times x_1 + b_2 \times x_2 + b_3 \times x_3 + b_{11} \times x_1^2 + b_{22} \times x_2^2 + b_{33} \times x_3^2 + b_{12} \times x_1 \times x_2 + b_{13} \times x_1 \times x_3 + b_{23} \times x_2 \times x_3 \quad (2)$$

Where b₀ is constant, b₁, b₂ and b₃ are linear coefficients of SCN concentration (x₁), H₂O₂ concentration (x₂) and nisin concentration (x₃), b₁₂, b₁₃ and b₂₃ are cross product coefficients and b₁₁, b₂₂ and b₃₃ are

<table><caption>**Table 2**
Code and level of variables chosen for the trials.</caption><thead><tr><th rowspan="2">Factors</th><th colspan="2">Symbols</th><th colspan="5">Level<sup>a</sup></th></tr><tr><th>Coded</th><th>Uncoded</th><th>-1.68</th><th>-1</th><th>0</th><th>1</th><th>1.68</th></tr></thead><tbody><tr><td>Thiocyanate (mg/L)</td><td>x<sub>1</sub></td><td>X<sub>1</sub></td><td>2.24</td><td>7</td><td>14</td><td>21</td><td>25.76</td></tr><tr><td>Hydrogen peroxide (mg/L)</td><td>x<sub>2</sub></td><td>X<sub>2</sub></td><td>4.8</td><td>15</td><td>30</td><td>45</td><td>55.2</td></tr><tr><td>Nisin (IU/mL)</td><td>x<sub>3</sub></td><td>X<sub>3</sub></td><td>3.63</td><td>10</td><td>30</td><td>50</td><td>63.63</td></tr></tbody></table>

<footnote>a x₁ = (X₁ - 14)/7; x₂ = (X₂ - 30)/15; x₃ = (X₃ - 30)/20.</footnote>
<caption>**Table 3**</caption>

Different concentrations of thiocyanate, hydrogen peroxide and nisin used to study the impact of lactoperoxidase activation and nisin on keeping the quality of raw milk.

<table><thead><tr><th>N°</th><th>Thiocyanate (mg/L)</th><th>H<sub>2</sub>O<sub>2</sub> (mg/L)</th><th>Nisin (IU/mL)</th></tr></thead><tbody><tr><td>1</td><td>21</td><td>45</td><td>10</td></tr><tr><td>2</td><td>21</td><td>45</td><td>50</td></tr><tr><td>3</td><td>14</td><td>30</td><td>30</td></tr><tr><td>4</td><td>14</td><td>30</td><td>30</td></tr><tr><td>5</td><td>14</td><td>30</td><td>30</td></tr><tr><td>6</td><td>14</td><td>4.8</td><td>30</td></tr><tr><td>7</td><td>7</td><td>15</td><td>10</td></tr><tr><td>8</td><td>21</td><td>15</td><td>10</td></tr><tr><td>9</td><td>14</td><td>30</td><td>3.6</td></tr><tr><td>10</td><td>21</td><td>15</td><td>50</td></tr><tr><td>11</td><td>25.76</td><td>30</td><td>30</td></tr><tr><td>12</td><td>2.24</td><td>30</td><td>30</td></tr><tr><td>13</td><td>7</td><td>15</td><td>50</td></tr><tr><td>14</td><td>7</td><td>45</td><td>10</td></tr><tr><td>15</td><td>14</td><td>30</td><td>63</td></tr><tr><td>16</td><td>14</td><td>55.2</td><td>30</td></tr><tr><td>17</td><td>7</td><td>45</td><td>50</td></tr></tbody></table>

quadratic coefficient. The obtained fit of second order equation was checked by the coefficient of determination R² and R² adjusted. The analysis of variance and the estimation of response surface by multiple linear regressions were performed using the software STATGRAPHICS Centurion XV version 15.2.06.

## 2.6. Study of the effect of temperature and lactoperoxidase activation

To study the effect of temperature and LPS activation, twelve experiments were carried out (Table 3). The used heat treatment scale corresponded to pasteurization (63 °C for 30 min). Microbiological analysis was carried out before and after each treatment. Thus, samples were periodically withdrawn (with an interval of 4 days), to estimate the total bacterial count, coliforms, yeasts and molds.

## 2.7. Statistical analysis

Experiments were repeated three times. Differences between means were estimated using SPSS version software (SPSS, Chicago, IL). Statistical significance for differences was determined at a 5 % probability level.

# 3. Results

## 3.1. Effect of LPS activation and the addition of nisin on the microbiological quality of milk

### 3.1.1. Predictive response model

The combined effect of LPS activation (by the addition of SCN and H₂O₂) and nisin at 25 °C for 72 h, on the microbiological quality of raw milk was studied using response surface methodology (RSM). The responses (Y) measured in terms of log-cycle reduction (log N₀/N) of total flora (Y₁), coliforms (Y₂) and yeasts and molds (Y₃) counts are given in Table 4. Each response was represented by a second-order polynomial equation containing 10 estimated coefficients.

The analysis of variance (ANOVA) of the quadratic model, realized by STATGRAPHICS software, indicated that the R² and R² adjusted values, being close to 1, illustrated a good fit of the derived model to the experimental data. The Fisher F-test and P values were used as a tool to check the significance of each coefficient indicating the pattern of interactions between variables. Obtained results showed differences in the meaning of each coefficient across different equations. For example, the linear coefficients (x₁, x₂ and x₃) were significant with low probability values (p < 0.05) across all equations. In the case of coliforms, only the quadratic coefficient (x²) was not significant. For total mesophilic

<caption>**Table 4**</caption>

Different conditions used to study the effect of temperature and activation of lactoperoxidase system on microbiological quality of raw cow milk.

<table><thead><tr><th>No. of experiments</th><th>Conditions</th><th>Incubation Temperature</th></tr></thead><tbody><tr><td>1</td><td>Control (without processing and activation)</td><td>These experiments were carried out at 25 °C and 4 °C</td></tr><tr><td>2</td><td>Heat treatment (63 °C for 30 min) (TT)</td><td></td></tr><tr><td>3</td><td>Activation of lactoperoxidase system (A)</td><td></td></tr><tr><td>4</td><td>Heat treatment after 2 h of LPS activation (A2+TT)</td><td></td></tr><tr><td>5</td><td>Heat treatment after 4 h of LPS activation (A4+TT)</td><td></td></tr><tr><td>6</td><td>Heat treatment after 6 h of LPS activation (A6+TT)</td><td></td></tr></tbody></table>

counts and molds, in addition to the linear coefficients, only the cross coefficients (x₂×₃) and quadratic (x²) were not significant, respectively.

### 3.1.2. Response surfaces

The total bacterial count, coliforms, yeasts and molds are indicative of a non-sterility issue in raw cow milk. Indeed, their maximum levels in raw milk are approximately the order of 10⁶, 10⁵ and 10⁴ CFU/mL, respectively. Therefore, reductions of 4log, 5log and 6log from the initial number of yeasts and molds, coliforms and total mesophilic bacteria were targeted of their inactivation through combined treatment of nisin with LPS activation for 72 h at 25 °C. The response surfaces of the regression equations were generated using STATGRAPHICS and are presented in Figs. 1–3. In each figure, one factor is held constant at its optimal value as determined by software.

#### 3.1.2.1. Total mesophilic bacteria.
The interaction of H₂O₂ and nisin concentrations at a constant SCN concentration (7.1 mg/L) is illustrated in Fig. 1a. The reduction in initial number of total bacterial count increases with increasing nisin concentration and decreasing of H₂O₂ concentration. It reached 6log at concentrations of around 50 IU/mL of nisin and 15 mg/L of H₂O₂.

Fig. 1b shows the inactivation rate of the total bacterial count as a function of SCN and nisin concentrations in the presence of 15.23 mg/L of H₂O₂. In fact, 6log and 3log reductions were observed at SCN concentration lower than 9 mg/L and after incubation of less than 50 h at 4 °C and 25 °C, respectively. Indeed, a 6log reduction in the initial number of bacteria was obtained at concentrations of 7 mg/L of SCN and 50 IU/mL of nisin.

The effect of SCN and H₂O₂ concentrations, while maintaining nisin at its optimum value (49 UI/mL), on the destruction of total germs showed that the inhibition rate increases as SCN and H₂O₂ concentrations decrease (Fig 1c). Indeed, a 6log reduction in the initial counts of total bacterial count was achieved in the presence of SCN and H₂O₂ concentrations between 7 and 8 mg /L and 15–18 mg/L, respectively.

#### 3.1.2.2. Coliforms.
The interaction of nisin and H₂O₂ at a concentration around 7 mg/L of SCN is illustrated in Fig. 2a. At nisin concentrations above 35 IU/mL and H₂O₂ concentrations below than 15 mg/L, a reduction of up to 3.5log in the initial number of coliforms was observed.

The reduction of coliforms counts when nisin and SCN were added at a fixed concentration of H₂O₂ (15 mg/L) is shown in Fig. 2b. A reduction greater than 3.5log was achieved at nisin concentrations greater than 35 IU/ mL and SCN concentrations less than 9 mg /L.

Fig. 2c demonstrates the effect of SCN and H₂O₂ concentrations on the reduction of coliforms while keeping nisin constant at its optimum value (50UI/mL). This figure shows that the reduction of the initial count of coliforms increases with decreasing SCN and H₂O₂ concentrations, reaching 5log reduction in the presence of 15 mg/L and 22 mg/L
a

Thiocyanate (7.1 mg/L)

<figure><img src="image_2.png" /></figure>

b

Hydrogen peroxide (15.23 mg/L)

<figure><img src="image_3.png" /></figure>

c

Nisin (49 UI/mL)

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Response surface curves showing the effect of nisin and hydrogen peroxide (a), nisin and thiocyanate (b), and thiocyanate and hydrogen peroxide (c) on the destruction of mesophilic total bacteria in raw cow milk at 25 °C.</figcaption></figure>

of SCN and H₂O₂, respectively.

3.1.2.3. *Yeasts and molds.* The effect of H₂O₂ and nisin, at a constant concentration of SCN (7.03 mg /l), is shown in Fig. 3a. A 4log reduction was observed at concentrations of approximately 50 IU/mL for nisin and 15 mg/mL for H₂O₂.

The reduction of yeasts and molds as a function of nisin and SCN concentrations, at a fixed H₂O₂ concentration (15.35 mg/mL) is shown in Fig. 3b. A 4log reduction was achieved at 25 °C for 72 h, in the presence of 7 mg/L of H₂O₂ and 50 IU/mL of nisin.

Fig. 3c illustrates the effect of SCN and H₂O₂ concentrations while

keeping the concentration of nisin to its optimum value (49.5 UI/mL) on the destruction of yeasts and molds at 25 °C for 72 h. A 4log reduction was obtained in the presence approximately of 8 mg/L of SCN and 16 mg/L of H₂O₂ concentration.

### 3.1.3. *Models validation*

The used models were verified by conducting ten different experiments using combinations of thiocyanate, hydrogen peroxide, and nisin combinations at various parameters. The experimental values closely matched the predicted values with a high correlation coefficient (R²) of 0.99 and a statistical significance level of *P* < 0.0001. Therefore, the
<figure><img src="image_5.png" /><figcaption>Fig. 2. Response surface curves showing the effect of nisin and hydrogen peroxide (a), nisin and thiocyanate (b), and thiocyanate and hydrogen peroxide (c) on the destruction of coliforms in raw cow milk at 25 °C.</figcaption></figure>

model can be considered as quite a reliable method for predicting the effect of LPS activation and nisin on the quality of raw milk.

## 3.2. Study of the effect of lactoperoxidase activation in combination with heat treatment on the quality of raw milk

Within this work, we contributed to improve the microbiological quality of raw milk using heat treatment combined with LPS activation.

LPS was achieved through the addition of SCN and $H_2O_2$. The control of the microbiological quality of raw milk was monitored by assessing the total bacterial count, total coliforms, yeasts and molds before and after each treatment at two different temperatures (4 °C and 25 °C). Thus, the obtained results are represented as the mean of $log_{10}$ (CFU/mL).

### 3.2.1. Case of total bacterial count

Fig. 4 illustrates the evaluation of total bacterial count in raw milk
<figure><img src="image_6.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 3. Response surface curves showing the effect of nisin and hydrogen peroxide (a), nisin and thiocyanate (b), and thiocyanate and hydrogen peroxide (c) on the destruction of yeasts and molds in raw cow milk at 25 °C.</figcaption></figure>

under different treatments (control, heat treatment alone, LPS-
activation alone and heat treatment-activation) at 4 °C and at 25 °C
for 96 h. Indeed, the analysis of the growth kinetics of these germs in the
control sample (without treatment) shows a continuous increase over
time reaching over 10⁶ CFU/mL at 4 °C and 10⁷ CFU/mL at 25 °C, after
96 h of incubation. Following heat treatment (63 °C for 30 min), a sig-
nificant reduction in this count is noted, with a halving observed after 24
h at 25 °C. In fact, a reduction of approximately 4.4 log at 4 °C was
observed. However, a slight increase was noted after 48 h of treatment
reaching 3.6log and 4.7log at 4 °C and 25 °C after 96 h, respectively.
Following LPS activation (by adding SCN and H₂O₂), the number of total

germs fluctuated over 24 h, corresponding to 2log at 4 °C and 3.9log at
25 °C. Thereafter, a progressive increase was noticed after 48 h, 72 h and
96 h and reached 3.5log at 4 °C and 25 °C. The curves summarizing the
combined effect of LPS activation for 2 h, 4 h and 6 h with heat treat-
ment show that this combination had a very significant antimicrobial
effect. However, the efficacy depends on both the duration of LPS acti-
vation and the incubation temperature. Indeed, with LPS activation for 2
h followed by a heat treatment, the total bacterial count decreased
significantly by a value of 2log resulting in a reduction of 2.19log at 4 °C
and 1.7log at 25 °C after 96 h. Thus, activating LPS for 4 h followed by a
heat treatment resulted in a gradual reduction of total germs load
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Evolution of total mesophilic bacteria in raw cow milk according to different treatments at 4 °C (A) and 25 °C (B) for 96 h.</figcaption></figure>

reaching a reduction of approximately 3.24 log at 4 °C and 2.2 log at 25 °C after 96 h. Finally, activating LPS for 6 h with the heat treatment gave a significant reduction in the initial number of total germs reaching up to 5 log after 96 h at 4 °C.

### 3.2.2. Case of total coliforms

Fig. 5 summarizes the number of total coliforms in raw milk, after various treatments, incubated at 4 °C and 25 °C for 96 h. As indicated in results, the bacterial growth in raw milk gradually increased from 3.4 log to 4.1 log at 4 °C and 5.6 log at 25 °C, after incubation for 96 h. However, following heat treatment and/or LPS activation, this growth was significantly reduced. Indeed, a total reduction of these germs in raw milk was achieved after LPS activation for 4 or 6 h followed by heat treatment, incubated for 96 h at 4 °C. However, this strong antimicrobial effect of LPS and temperature was not observed when milk was incubated at 25 °C. In fact, the important reduction (2.1 log) was observed after activating LPS for 6 h followed by heat treatment at 25 °C for 96 h.

### 3.2.3. Case of yeasts and molds

The effect of LPS activation, with and without the heat treatment, on the destruction of yeasts and molds in raw milk at 4 °C and 25 °C for 96 h is illustrated in Fig. 6. It is evident that the growth rate of these germs in the absence of treatment (control) is greater at 4 °C than at 25 °C. Specially, the yeast and molds count increased from 3.5 log to 3.9 log at 4 °C and to 5.6 log at 25 °C after 96 h of incubation. In addition, LPS activation or the application of heat treatment alone generated a transient reduction in microbial load observed after 24 h of incubation, followed by a slight increase, reaching 3.7 log at 25 °C and 2.7 log at 4 °C after 96 h of heat treatment. However, when a heat treatment was

preceded by LPS activation, the inactivation rate of these germs increased significantly at both 4 °C and 25 °C, reaching complete destruction at 4 °C and a reduction of around 2 log at 25 °C after 96 h.

## 4. Discussion

The optimal values of each parameter (SCN, H₂O₂ and nisin) to achieve a reduction of 6 log, 5 log and 4 log of the initial number of total bacteria, coliforms and yeasts and molds were approximately 7 mg/L of thiocyanate, 15 mg/L of H₂O₂, and 50 IU/mL of nisin, obtained after 72 h of incubation at 25 °C (Table 5). The results demonstrated that the antimicrobial effect of LPS activation and nisin varied among the studied microorganisms (total bacterial count, coliforms, yeasts and molds). Furthermore, the reduction rate of various microorganisms increases with decreasing SCN and H₂O₂ concentrations, and increasing nisin concentration in raw milk incubated at 25 °C for 72 h.

Based on the obtained data, we observe that the doses required to inactivate bacteria in raw milk are lower than those of international standards (14 mg/L of the thiocyanate and 30 mg/L of H₂O₂) and differ from those found in other studies. This discrepancy may be attributed to the synergistic effect of LPS and nisin. For instance, according to Fanta et al. (2019), concentrations of 14 mg/L of SCN and 20 mg/L of H₂O₂ were utilized to activate LPS in cow milk collected from Ethiopia. In addition, Boulares et al. (2011) demonstrated that concentrations of 28 mg/L of SCN and 60 mg/L of H₂O₂ were effective in preserving the Tunisia raw milk at 4 °C for 72 h. Ahmed et al. (2021) specified that LPS was activated by dissolving 40 mg of SCN and 30 mg of sodium percarbonate, respectively. Furthermore, LPS was shown to reduce viable bacterial count for up 6 h of incubation and preserve milk from clotting
<figure><img src="image_10.png" /><figcaption>Fig. 5. Evolution of total coliforms in raw cow milk according to different treatments at 4 °C (A) and 25 °C (B) for 96 h.</figcaption></figure>

for at least 4 h of incubation (Ahmed et al., 2021). The differences noted among these reports could be related to the initial quality of milk (Lara-Aguilar & Alcaine, 2019; Silva et al., 2020).

Concerning the effect of LPS activation followed by heat treatment on the inhibition of total bacterial count, coliforms, yeasts and molds, we observed that the antimicrobial effect of LPS varies depending on the micro-organisms, the duration of LPS activation (2 h, 4 h and 6 h) and the incubation temperature (4 °C and 25 °C). In fact, its effect more pronounced at 4 °C than at 25 °C. In addition, the reduction rate in the initial number of different germs at 4 °C and 25 °C increases with longer duration of LPS activation in raw milk. Indeed, a complete destruction of coliforms, yeasts and molds was observed, at 4 °C for 96 h, following LPS activation for 4 h and 6 h, followed by heat treatment.

The synergistic effect observed between LPS-activation and either nisin or heat treatment can be explained by two hypotheses. Firstly, lactoperoxidase may disrupt cells function by targeting their cytoplasmic membranes which serve as the permeability barrier, thereby increasing susceptibility to nisin. Secondly, treatment with antimicrobial agents or heat may weakens the cell wall, thereby reducing the sensitivity of organisms that are typically resistant to the antimicrobial effect of the LPS. In fact, Boussouel et al. (2000), tested the individual or combined effects of nisin (100 or 200 IU/mL) and LPS against L. monocytogenes ATCC 15,313 in milk at 25 °C over 15 days. These researchers showed that nisin application had bactericidal effect while LPS showed bacteriostatic effect. In both cases, regrowth of L. monocytogenes was observed. However, combining LPS and nisin resulted in a synergic bactericidal effect. Indeed, after 8 days, no bacteria were detected in 1 mL of milk (Boussouel et al., 2000). Furthermore, Elotmani and Assobhei (2003) investigated the antimicrobial effects of nisin and LPS

on fish microflora. They found that nisin alone inhibited Gram-positive bacteria, whereas, LPS inhibited all tested strains. The combination of nisin (100 IU/mL) and LPS was significantly more effective than either LPS or nisin alone against all studied strains. In contrast, Khudhir and Hammad (2016) revealed that *E. coli* O157:H7 was resistant to nisin. When nisin was employed with activated LPS, a significant increase in *E. coli* O157:H7 counts was observed. A study conducted in 2004 reported that heat treatment is more effective when the initial number of cells is minimized before treatment. Therefore, applying LPS before heating ensures a complementary, and even synergistic, combination (CAC, 2004). Generally, the antibacterial activity of LPS is attributed to the production of hypothiocyanite through the enzymatic reaction between H₂O₂ and SCN. Hypothiocyanite is a short-lived product that kills bacteria, fungi, and viruses by disrupting the sulfhydryl (SH) groups of the cell membrane (Bafort et al., 2014).

From the analysis of all the obtained data, it can be concluded that the inhibition of coliforms is more significant compared to that of total bacterial count, and even compared to yeasts and molds, which confirms the bactericidal effect of the LPS against Gram-negative bacteria is greater than against Gram-positive ones. Sarr et al. (2018) showed that these differences can be explained by variations in the cell wall structure and properties.

## 5. Conclusion

The present work investigated the effect of LPS activation, nisin, or heat treatment on the microbiological quality of raw milk incubated at 25 °C for 72 h. Initially, the results demonstrated that LPS and nisin acts synergistically, resulting in a very significant inhibitory effect on the
<figure><img src="image_11.png" /><figcaption>Fig. 6. Evolution of yeasts and molds in raw cow milk according to different treatments at 4 °C (A) and 25 °C (B) for 96 h.</figcaption></figure>

<caption>**Table 5**</caption>

Different equations and their correlation coefficients obtained in the inactivation of total mesophilic bacteria, coliform bacteria, yeasts and molds at 25 °C after activation of LPS-system and addition of nisin.

<table><thead><tr><th>Microorganisms</th><th>Equations (Y= log N<sub>0</sub>/N)</th><th>R<sup>2</sup> (Correlation coefficient)</th><th>R<sup>2</sup> adjusted</th></tr></thead><tbody><tr><td>Total bacterial count</td><td>Y<sub>2</sub> = 4.47 + 0.07×<sub>1</sub> - 0.08×<sub>2</sub> + 0.02×<sub>3</sub> + 0.006×<sub>1</sub><sup>2</sup> + 0.006×<sub>1</sub>×<sub>2</sub> - 0.003×<sub>1</sub>×<sub>2</sub> - 0.001×<sub>2</sub><sup>2</sup> + 0.0001×<sub>2</sub>×<sub>3</sub> + 0.0008×<sub>3</sub><sup>2</sup></td><td>0.989</td><td>0.972</td></tr><tr><td>Coliforms</td><td>Y<sub>1</sub> = 2.82 + 0.03×<sub>1</sub> - 0.12×<sub>2</sub> + 0.08×<sub>3</sub> - 0.004×<sub>1</sub><sup>2</sup> + 0.007×<sub>1</sub>×<sub>2</sub> - 0.005×<sub>1</sub>×<sub>3</sub> - 0.0007×<sub>2</sub><sup>2</sup> - 0.001×<sub>2</sub>×<sub>3</sub> + 0.0007×<sub>3</sub><sup>2</sup></td><td>0.989</td><td>0.981</td></tr><tr><td>Yeasts and molds</td><td>Y<sub>3</sub> = 5.77 - 0.26×<sub>1</sub> - 0.31×<sub>2</sub> + 0.06×<sub>3</sub> + 0.002×<sub>1</sub><sup>2</sup> + 0.006×<sub>1</sub>×<sub>3</sub> - 0.002×<sub>1</sub>×<sub>3</sub> + 0.003×<sub>2</sub><sup>2</sup> - 0.001×<sub>2</sub>×<sub>3</sub> + 0.0009×<sub>3</sub><sup>2</sup></td><td>0.989</td><td>0.968</td></tr></tbody></table>

x₁ is thiocyanate concentration, x₂ is hydrogen peroxide concentration and x₃ is nisin concentration.

microflora of raw milk (total bacteria, coliform bacteria, and yeasts and molds). However, this antimicrobial effect varied slightly depending on the microorganism. The optimal values for each parameter (SCN, H₂O₂ and nisin) to achieve reduction of 6log, 5log and 4log in the initial number of total bacteria count, coliforms, yeasts and molds were

approximately 7 mg / L of SCN, 15 mg / L of H₂O₂ and 50 IU/mL of nisin, obtained after 72 h of incubation at 25 °C. Notably, the doses of SCN and H₂O₂ required for LPS activation were lower than international standards, highlighting the potential of experimental design in determining the adequate concentrations for LPS activation in raw milk. Similarly, the effectiveness of inactivating various germs in raw milk by LPS activation in combination with heat treatment (63 °C for 30 min) is closely related to the duration of activation before heat treatment and the incubation temperature. Complete destruction of coliforms, yeasts and molds was observed at 25 °C for 96 h following LPS activation for 4 h and 6 h, followed by heat treatment (63 °C for 30 min). These results suggest future possibilities for potential applications of this system to enhance the microbiological quality of other dairy products.

## Ethical statement - Studies in humans and animals

During this work, we don't have a studies in humans and animals.

## CRediT authorship contribution statement

**Chedia Aouadhi:** Writing – original draft, Visualization, Supervision, Software, Investigation, Data curation, Conceptualization. **Souhir Kmiha:** Writing – review & editing, Methodology, Formal analysis. **Faten Trakhna:** Writing – review & editing, Methodology, Formal analysis. **Abderrazak Maaroufi:** Writing – review & editing, Validation, Project administration, Funding acquisition.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.

Acknowledgment

The authors acknowledge the financial support provided by Tunisian Ministry of Higher Education and Scientific Research (LR16IPT03).

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