Inhibitory effect of licorice extract on the germination and outgrowth of Paraclostridium bifermentans spores

Abstract

Introduction: Paraclostridium bifermentans is responsible for spoilage properties in vacuum-packaged meat. Ordinary heat treatment techniques are ineffective to control the extremely heat-resistant spores of P. bifermentans. Therefore, finding a new strategy to prevent the contamination of P. bifermentans spores in vacuum-packaged meat is challenging.

Methods: In this study, P. bifermentans was isolated from the vacuum-packaged chicken, and the inhibitory effects of licorice extract on the germination and outgrowth of P. bifermentans spores, as well as the key bioactive components in the licorice extract involved in inhibiting spore activity, were investigated.

Results: The spores induced by combination-nutrient-germinant (150 mmol/L L-alanine and 20 mmol/L inosine, co-AI) did not germinate when the concentration of licorice extract was ≥ 3.13 mg/ml. The germination of P. bifermentans spores induced by non-nutrient-germinant (8 mmol/L dipicolinic acid, DPA) was completely prevented by licorice extract at least 1.56 mg/ml. While the outgrowth of P. bifermentans spores was inhibited at a concentration of 0.39 mg/ml. Licorice extract did not seem to damage the non-germinated spores but blocked the germinant sensing. Licorice extract prevented the outgrowing spores from becoming vegetable cells by disrupting the inner membrane. Furthermore, the results obtained from LC-MS data analysis exhibited 15 key bioactive compounds in licorice extract, such as glycyrrhizic acid, liquiritin, etc. Among them, glycyrrhizic acid and liquiritin apioside exerted efficient inhibitory properties on the germination and outgrowth of P. bifermentans spores.

Discussion: This present study demonstrated that licorice extract can be used as a promising inhibitor of spores and provides a new method to control the residual P. bifermentans spores in meat products. Meanwhile, this study exhibits a baseline for the better understanding of the potential application of licorice extracts to control the P. bifermentans spores in meat products.

Keywords: Paraclostridium bifermentans; germination; inhibition; licorice extract; outgrowth; spores.

Figure 1

Design of experiments. The licorice extract was obtained from the root of licorice (Glycyrrhiza uralensis Fisch), and its main bioactive compounds were detected by LC-MS. P. bifermentans was isolated from the vacuum-packaged chicken breast with “blown pack” spoilage and cultured under anaerobic conditions to obtain the spores. The experiment was divided into two parts, on the one hand, the ability of licorice extract to directly inhibit the initiation of spore germination was determined, and on the other hand, the effect of licorice extract to prevent the outgrowth of germinated spores was investigated. Subsequently, the pattern of the germination and outgrowth inhibited by licorice extracts was explored. Finally, the contributions of the five bioactive compounds identified from licorice extracts in the inhibition of germination and outgrowth were measured.

Figure 2

Inhibitory effect of licorice extract on the germination of P. bifermentans spores. (A) Inhibition of licorice extract on the germination of P. bifermentans spores in the presence of the combination-nutrient-germinant (150 mmol/L L-alanine and 20 mmol/L inosine, co-AI) and non-nutrient-germinant (8 mmol/L dipicolinic acid, DPA); Germination curves of P. bifermentans spores when treated with licorice extract at the minimum inhibitory spore germination concentration (MIGC, 3.13 mg/ml), 2 × MIGC (6.25 mg/ml), 1/2 × MIGC (1.56 mg/ml), and 0 × MIGC (0 mg/ml) in the presence of co-AI (B) and at 1 × MIGC (1.56 mg/ml), 2 × MIGC (3.13 mg/ml), 1/2 × MIGC (0.78 mg/ml), and 0 × MIGC (0 mg/ml) in the presence of DPA (C); (D) Effect of licorice extract on the thermal inactivation of P. bifermentans spores; PI staining results of P. bifermentans spores when treated with licorice extract at 0 mg/ml (E₁), 3.13 mg/ml (E₂) in the presence of co-AI and at 0 mg/ml (E₃), 3.13 mg/ml (E₄) in the presence of DPA; (F) The survival rate of P. bifermentans spores after treatment with licorice extract at corresponding MIGC in the presence of co-AI or DPA. Values with different uppercase letters were significantly different (p < 0.05) in the co-AI-induced group and values with different lowercase letters were significantly different (p < 0.05) in the DPA-induced group (A,D,F). Values with different uppercase letters were significantly different (p < 0.05) at the same time point in different concentration groups (B,C). The error bars represent standard deviations of the mean (n = 3).

Figure 3

Schematic model of the P. bifermentans spore germination inhibited by licorice extract. Germinants (co-AI, 150 mmol/L L-alanine, and 20 mmol/L inosine; DPA, 8 mmol/Ldipicolinic acid) are sensed by interactions with germination receptors. Subsequently, the germination of spores begins and is accompanied by the hydrolysis of the cortex and leakage of DPA. However, the binding of the germinants to the receptor was affected by licorice extract, resulting in the no occurrence of germinant sensing. Similar situations happen to glycyrrhizic acid and liquiritin apioside, which contain more hydroxyl or carboxyl group, expanding their ability to prevent the reaction between the germinants and the receptors. As to liquiritin, licoisoflavone A, and liquiritigenin have relatively few hydroxyl groups, they are unable to completely inhibit the germinant sensing, and the spores will germinate partially.

Figure 4

Inhibitory effect of licorice extract on the outgrowth of P. bifermentans spores. (A) Inhibitory of licorice extract on the outgrowth of P. bifermentans spores induced by the combination-nutrient-germinant (150 mmol/L L-alanine and 20 mmol/L inosine, co-AI) and non-nutrient-germinant (8 mmol/L dipicolinic acid, DPA); Outgrowth curves of P. bifermentans spores induced by co-AI (B) or DPA (C) when treated with licorice extract at the minimum inhibitory spore outgrowth concentration (MIOC, 0.39 mg/ml), 2 × MIGC (0.78 mg/ml), 1/2 × MIGC (0.19 mg/ml), and 0 × MIGC (0 mg/ml); PI staining results of P. bifermentans spores induced by co-AI when treated with licorice extract at 0 mg/ml (D₁), 0.39 mg/ml (D₂) for 3 h and at 0 mg/ml (D₃), 0.39 mg/ml (D₄) for 6 h or induced by DPA when treated with licorice extract at 0 mg/ml (D₅), 0.39 mg/ml (D₆) for 3 h and at 0 mg/ml (D₇), 0.39 mg/ml (D₈) for 6 h; (E) The survival rate of P. bifermentans spores induced by co-AI or DPA when treated with licorice extract at MIOC for 3 h and 6 h; (F) Effects of licorice extract on the oxidative metabolism of P. bifermentans spores induced by co-AI or DPA; (G) Leakage of DNA and RNA from P. bifermentans spores induced by co-AI or DPA when treated with licorice extract at MIOC. Values with different uppercase letters were significantly different (p < 0.05) in the co-AI-induced group and values with different lowercase letters were significantly different (p < 0.05) in the DPA-induced group (A,E,F). Values with different uppercase letters were significantly different (p < 0.05) at the same time point in different concentration groups (B,C). Values with different uppercase letters were significantly different (p < 0.05) in the OD₂₆₀ group and values with different lowercase letters were significantly different (p < 0.05) in the OD₂₈₀ group (G). The error bars represent standard deviations of the mean (n = 3).

Figure 5

Schematic model of the P. bifermentans spore outgrowth inhibited by licorice extract. The spore inner membranes are more accessible for licorice extract due to the hydrolysis of the cortex and the abscission of the coat, causing the leakage of contents, the cessation of oxidative metabolism, and the entry of PI. On the contrary, no PI can enter the complete membranes, and metabolism and synthesis proceeded normally when licorice extract is absent.

Figure 6

Schematic model for licorice extract to inhibit the germination and outgrowth of P. bifermentans spores. The non-germinated spores will be triggered by germinants such as L-alanine, inosine, and dipicolinic acid (DPA). On the one hand, prior to germination, the addition of licorice extract (glycyrrhizic acid, liquiritin apioside, etc.) will prevent the binding of germinants to receptors, resulting in the no occurrence of germinant sensing. On the other hand, the outgrowing spores, upon the hydrolysis of the cortex, the release of DPA, and the abscission of the coat, lose their resistance. Treatment with licorice extract at this point will cause damage to the inner membrane of the outgrowing spores and prevent them from becoming vegetative cells.