Development of Alginate Capsules with Bioactive Compounds from Lactobacillus plantarum GP108 and Evaluation of Their Effect Against Escherichia coli ^†

Abstract

Microbial growth in food represents a public health problem that requires immediate attention. In Ecuador, 8924 cases of foodborne illness (FBD) were reported in 2020, most of them caused by bacteria. It is estimated that Escherichia coli causes 2,801,000 acute illnesses per year and results in 3890 cases of hemolytic uremic syndrome, 270 cases of end-stage renal disease and 230 deaths. Under this context, in this work, alginate capsules containing bioactive compounds from Lactobacillus plantarum GP108 were developed and their antimicrobial effect against E.coli was measured. The formulation of the capsules was carried out using a completely randomized experimental design with three formulations: maximum, average and minimum. The antimicrobial activity was measured by in vitro tests based on the increase in optical density during 7 days of exposure of E. coli with the capsules. By analysis of variance (ANOVA), it was found that the percentage inhibition of the capsules depended only on the formulation (p-value < 0.05), but not on the number of exposure capsules (p-value > 0.05). Tukey’s test indicated that the average formulation is the best at inhibiting the growth of E. coli, maintaining an average of 11.66% inhibition for 7 days. These findings show that bioactive compounds produced by L. plantarum GP108 encapsulated in alginate could be of potential use for food biopreservation.

1. Introduction

Microbial growth in food represents a public health problem. According to the National Directorate of Epidemiological Surveillance of Ecuador, 8924 cases of foodborne diseases were reported in 2020. Of this figure, 66% were intoxications produced by bacteria [1].

In general, bacteria are the main cause of FBDs. Escherichia coli is the pathogen with the highest incidence in this type of disease. This bacterium represents a very high health risk and is an indicator of fecal contamination in food [1]. It is estimated that E. coli causes 2,801,000 acute illnesses per year and results in 3890 cases of hemolytic uremic syndrome, 270 cases of end-stage renal disease, and 230 deaths [2].

In recent years, there has been a growing interest in the use of microorganisms or their metabolites to extend shelf life and maintain food safety. Thus, biopreservation by encapsulation of bioactive compounds has emerged as an innovative alternative [3]. Numerous studies have confirmed the efficacy of this method, finding very promising results for the food industry [3,4]. Under this context, this work focused on developing alginate capsules with bioactive compounds from L. plantarum GP108 with an antimicrobial effect against Escherichia coli.

2. Materials and Methods

2.1. Obtaining the Substrate with Bioactive Compounds (SB)

We proceeded according to Bravo-Pulla [5], with slight modifications. L. plantarum GP108 (isolated from artisanal cheeses produced in Austro, Ecuador) was grown in Man Rogosa Sharpe (MRS) medium (Acumedia) at pH 5.5, incubated at 35 ± 1 °C in anaerobiosis for 72 h (Esco Isotherm Incubator, Esco Lifesciences, Horsham, PA, USA). Subsequently, the culture obtained was transferred to a flask with MRS broth (Acumedia Manufacture, Lansing, MI, USA) and a control was prepared. The inoculated flask and the control were incubated under the conditions described above.

SB was prepared as described by Bravo-Pulla [5]. The L. plantarum GP108 suspension was centrifuged (Eppendorf Centrifuge 5804, Eppendorf, Hamburg, Germany) and the cell-free supernatant (SLC) was adjusted to pH 6. Subsequently, the SLC was passed through 0.22 µm surfactant-free cellulose acetate filters (Thermo Scientific Nalgene Syringe Filter, Thermo Scientific, Waltham, MA, USA) and concentrated by evaporation at 45 ± 1 °C for 4 days.

2.2. Sensitivity Tests Against E. coli

According to Bravo-Pulla [5], a stock culture of E. coli ATCC 8739 was obtained in nutrient broth (36 ± 1 °C, 24 h) and adjusted to a concentration of 10⁸ CFU/mL by optical density (λ = 600 nm).

Sensitivity tests were performed by preparing 1:1 mixtures of SB and MRS with 10³ and 10⁵ CFU/mL E. coli bacterial suspensions. The mixtures were incubated under the same conditions as the stock culture. As described by Dey et al. [6], the initial (DO₀) and final OD (DO_f) were recorded at λ = 600 nm. The percentage of growth was calculated with Equation (1).

$${\mathrm{C}}_{\mathrm{\%}}={\displaystyle \frac{{\mathrm{D}\mathrm{O}}_{\mathrm{f}}-{\mathrm{D}\mathrm{O}}_0}{{\mathrm{D}\mathrm{O}}_{\mathrm{f}}}}\times 100$$

(1)

2.3. Preparation of Capsules with Bioactive Compounds (BCCs)

A completely randomized experimental design according to the criteria of Earle and McKee [7] was used to prepare the BCCs. Three formulations composed of two solutions were used (

Table 1. Formulation of Solution 1.

	Concentration (% w/w)
	Minimum	Average	Maximum
Alginate	1.2	1.8	2.4
Maltodextrin	10.8	10.2	9.6
Drinking water	78	73	68
SB/MRS	10	15	20
Glycerol	20 *	20 *	20 *

* Percentage with respect to the weight of alginate.

and

Table 2. Formulation of Solution 2.

	Concentration (% w/w)
	Minimum	Average	Maximum
CMC	0.76	0.75	0.74
Drinking water	93.7	92.6	91.6
CaCl2	5.55	6.61	7.66

). In addition, glycerol was used as a plasticizer, as described by Giz et al. [8].

For the formation of the capsules, we proceeded as described by Le et al. [4], with slight modifications. Ten milliliters of solution 1 was taken in a serological pipette and dropped dropwise into solution 2. The capsules were placed on a sterile paper towel for 5 min to remove excess liquid.

2.4. Sensitivity Testing of BCCs Against E. coli

Tubes of 10⁵ CFU/mL concentration of E. coli were prepared as described by Bravo-Pulla [5]. Subsequently, we proceeded according to Dey et al. [6], with some modifications. In detail, 1, 2, and 3 capsules per formulation, respectively, were placed in each tube, considering one sample for each day of measurement. They were incubated at 36 ± 1 °C for 7 days. The absorbance was measured at λ = 600 nm on days 0, 1, 4, 6, and 7. The samples were taken in triplicate and the same procedure was applied for the control tests. The results were expressed as percentage inhibition (I_%) using the following expression:

$${\mathrm{I}}_{\mathrm{\%}}={\displaystyle \frac{{\mathsf{\Delta }\mathrm{D}\mathrm{O}}_{\mathrm{C}}-{\mathsf{\Delta }\mathrm{D}\mathrm{O}}_{\mathrm{B}}}{{\mathsf{\Delta }\mathrm{D}\mathrm{O}}_{\mathrm{C}}}}\times 100$$

(2)

where ΔDO_C is the increase in optical density of the control samples in a given time interval, and ΔDO_B is the increase in optical density of samples containing bioactive compounds in the same time interval.

2.5. Statistical Analysis

For the statistical analysis of the data, an ANOVA and Tukey’s test were applied. Minitab 20 Statistical Software, version 20.3.0.0.0 and Excel 2019 were used.

3. Results and Discussion

3.1. SB Sensitivity Testing Against E. coli

Figure 1 shows the percentages of E. coli growth after 24 h of exposure to SB. SB is able to reduce 5.72 times the bacterial growth in 10⁵ CFU/mL suspensions of E. coli, presenting a bacteriostatic effect. Wang et al. [9] obtained a similar trend when exposing 10⁴ CFU/mL suspensions of E. coli to plantaricin BM-1. Likewise, Le et al. [4] reported a greater inhibitory effect of L. plantarum SC01 in bacterial suspensions of 10⁴ to 10⁸ CFU/mL.

In the 10³ CFU/mL suspension, no inhibitory effect was observed because E. coli was in the exponential phase of growth. As explained by Yates and Smotzer [10], at this stage, there is higher cell division and a higher nutrient concentration. In contrast, in suspensions of 10⁵ CFU/mL, E. coli enters the stationary phase, where nutrient availability and cell division decrease. On this basis, bioactive compounds show greater effectiveness at high bacterial loads due to the cellular vulnerability characteristic of E. coli growth kinetics.

3.2. Sensitivity Testing of BCCs Against E. coli

The inhibition percentages against E. coli for each BCC formulation can be seen in Figure 2. The maximum formulation BCCs showed no inhibitory effect (Figure 2a). For Kanipes et al. [11], CaCl₂ exerts a protective effect on E. coli. CaCl₂ induces the synthesis of phosphoethanolamine transferase: an enzyme capable of inactivating antimicrobial compounds. Likewise, other authors argue that high concentrations of Ca²⁺ ions produce a cross-linking of lipopolysaccharide molecules in Gram-negative bacteria, generating a more stable cell wall that prevents the action of antimicrobial compounds [12].

On the other hand, in the minimal formulation BCCs, although exposure to 1 as well as 3 capsules showed inhibition percentages, the inhibitory effect was barely seen on the fourth day (Figure 2c). This behavior could be attributed to the formulation of the capsules, a situation that will be analyzed later.

Finally, the exposure of E. coli to the average formulation BCCs showed an inhibitory effect from the first day. This behavior was the same when varying the number of capsules (Figure 2b). The percentage of inhibition is high in the first days and starts to decrease from the fourth day, showing a faster release of bioactive compounds at the beginning of the exposure. Niaz et al. [13] obtained a similar behavior in the release of nisin Z encapsulated in alginate and chitosan colloidosomes. Initially, the release is sudden and rapid. Later, it is slow and steady. The authors argue that this behavior is influenced by temperature and pH. Likewise, the release is higher at pH values close to neutrality than at acidic pH. Therefore, the sudden release of bioactive compounds in the average formulation BCCs can be explained by temperature and pH conditions. However, this can also be attributed to the formulation, as will be discussed later.

3.3. Analysis of Variance and Tukey’s Test

The analysis of variance (ANOVA) showed statistically significant differences in the percentage of inhibition when varying the formulation (p-value = 0.017). However, the same was not observed when varying the number of capsules in any formulation. Tukey’s test proves the above by demonstrating that the percentage of inhibition is independent of the number of capsules.

The average formulation has a significantly higher mean than the others. During the 7 days of exposure to the average formulation BCCs, an inhibition percentage of 11.66% is maintained. However, no significant difference or inhibitory effect was observed in the other formulations. Le et al. [4] evaluated the inhibitory activity of Lactobacillus plantarum SC01 encapsulated in alginate–gelatin. They reported an inhibition percentage of 65.87% in 10⁵ CFU/mL suspensions of E. coli after 48 h of exposure to the capsules. This gap can be attributed to the difference in the encapsulated material.

Several studies claim that the proportion of alginate influences the microstructure of the capsules and, therefore, the release of compounds [3,4,14]. Minimally formulated BCCs presented a delayed release of bioactive compounds due to the low alginate concentration (1.2%). At concentrations below 2%, irregular cross-linking of the polymer occurs. This generates stronger entrapment zones than others, limiting the availability and migration of the compounds [4].

On the other hand, the maximum formulation BCCs (2.4% alginate), being stiffer and denser, prevented the migration of the bioactive compounds to the surface. According to Narsaiah et al. [14], at values higher than 2% alginate, there are more binding sites for calcium, which increases cross-linking and forms a denser gel that hinders diffusion. Le et al. [4] reported the same behavior when the alginate concentration ranges between 2.5 and 3%.

As for the average formulation BCCs (1.8% alginate), it could be said that they presented a more uniform cross-linking. This proportion of alginate is close to that reported by Narsaiah et al. [14], who argue that at 2% alginate, optimal cross-linking occurs. For their part, authors such as Le et al. [4] found uniform cross-linking at an approximate concentration of 2.5% alginate. It is also important to consider that the properties of alginate can be complemented with the use of other polymers such as resistant starch [3], guar gum [14], or gelatin [4], making the proportion of alginate variable.

On the other hand, the properties of the capsules also depend on the CaCl₂ concentration and the use of plasticizers such as glycerol [8,15]. The high number of binding sites in the alginate and the high calcium concentration prevented the release of the bioactive compounds in the maximally formulated BCCs (cross-linked with 7.66% CaCl₂). According to studies, high calcium concentrations decrease the permeability of capsules [15]. The presence of several calcium ions produces a greater cross-linking of the alginate chains, hindering the diffusion of the compounds [8]. As for BCCs of minimal formulation (cross-linking with 5.55% CaCl₂), calcium cross-linking was not complete due to the irregular distribution of alginate binding sites [4].

The calcium concentrations used in this study are close to those reported in the literature. Hassan et al. [3] recommend a CaCl₂ concentration of 5% for optimal cross-linking. Likewise, Olivas and Barbosa-Canovas [15] found good cross-linking results using 10% CaCl₂ solutions. However, only BCCs of average formulation (6.61% CaCl₂) achieved a significant release of bioactive compounds. Therefore, it can be said that these capsules presented the best cross-linking. This could also be attributed to the interaction with glycerol within the formulation. Glycerol and calcium have a synergistic effect on the mechanical properties, permeability, and swelling of the capsules [15]. While calcium decreases permeability, glycerol increases it to some extent. Apparently, there is a point of equilibrium between calcium and glycerol for optimal cross-linking and thus adequate release of the encapsulated compounds [8].

4. Conclusions

Alginate capsules containing bioactive compounds with antimicrobial activity against E. coli were developed. The bioactive compounds showed activity before and after encapsulation. Therefore, the capsules were able to maintain their stability. Inhibition was found to be solely dependent on the formulation, with the formulation of 1.8% alginate, 15% SB, and 6.61% CaCl₂ being more effective, which managed to control E. coli growth for 7 days. These findings suggest that the encapsulation of bioactive compounds produced by L. plantarum GP108 represents a promising alternative for food biopreservation. However, further studies on its applicability, release kinetics, and characterization of the bioactive compounds are needed.