Strategies for effective high pressure germination or inactivation of Bacillus spores involving nisin

Abstract

The major challenge in employing high pressure (HP) at moderate temperature for sterilization is the remarkable resistance of bacterial spores. High isostatic pressure can initiate spore germination, enabling subsequent inactivation under mild conditions. However, not all spores could be triggered to germinate under pressure at temperatures ≤80°C so far. In this study, germination treatment combinations were evaluated for Bacillus spores involving moderate HP (150 MPa, 37°C, 5 min), very HP (vHP, 550 MPa, 60°C, 2.5 or 9 min), simple and complex nutrient germinants [L-valine, L-alanine, and tryptic soy broth (TSB)], nisin, and incubation at atmospheric pressure (37°C). The most effective combinations for Bacillus subtilis resulted in a reduction of culturable dormant spores by 8 log₁₀ units. The combinations involved nisin, a nutrient germinant (L-valine or TSB), a first vHP treatment (550 MPa, 60°C, 2.5 min), incubation at atmospheric pressure (37°C, 6 h), and a second vHP treatment (550 MPa, 60°C, 2.5 min). Such treatment combination with L-valine reduced Bacillus amyloliquefaciens spores by only 2 log₁₀ units. B. amyloliquefaciens, thus, proved to be substantially more HP-resistant compared to B. subtilis, validating previous studies. Despite combining different germination mechanisms, complete germination could not be achieved for either species. The natural bacteriocin nisin did seemingly not promote HP germination initiation under chosen HP conditions, contrary to previous literature. Nevertheless, nisin might be beneficial to inhibit the growth of HP-germinated or remaining ungerminated spores. Future germination experiments might consider that nisin could not be completely removed from spores by washing, thereby affecting plate count enumeration.

Importance: Extremely resistant spore-forming bacteria are widely distributed in nature. They infiltrate the food chain and processing environments, posing risks of spoilage and food safety. Traditional heat-intensive inactivation methods often negatively affect the product quality. HP germination-inactivation offers a potential solution for better preserving sensitive ingredients while inactivating spores. However, the presence of ungerminated (superdormant) spores hampers the strategy's success and safety. Knowledge of strategies to overcome resistance to HP germination is vital to progress mild spore control technologies. Our study contributes to the evaluation and development of mild preservation processes by evaluating strategies to enhance the HP germination-inactivation efficacy. Mild preservation processes can fulfill the consumers' demand for safe and minimally processed food.

Keywords: Bacillus amyloliquefaciens; Bacillus subtilis; endospore; germination; high isostatic pressure; inactivation; nisin; superdormant.

Fig 1

Graphical abstract of the experimental set-up of the HP spore germination–inactivation strategy and corresponding research questions. The experimental steps 2–5 were omitted or modified based on the research question. p: pressure; t: time.

Fig 2

Effect of the nisin concentration on vHP-treated or dormant (NoHP) B. subtilis spores, analyzed by flow cytometry. Spores were non-HP-treated or vHP-treated in buffer (pH 7.0) at 550 MPa and 60°C for 2.5 min, washed, and kept on ice until analysis. In regions of certain fluorescence signal heights (-H), the following spore subpopulations appear: PI- and SYTO16-negative ungerminated spores in region “R1”; SYTO16-positive germinated spores with an intact inner membrane in region “R2”; and PI-positive germinated and presumably inactivated spores with membrane damage in region “R3.” Note that events in R1 of vHP-treated samples are not ungerminated spores as their number is below the detection limit after this treatment time (27). Percentages of events per region are shown as the mean ± standard deviation of three experimental replicates. Nis: nisin.

Fig 3

Reduction of culturable dormant spores (N) after different germination treatments, washing, and heat treatment (80°C, 20 min), as analyzed by plate count. Dormant spores were treated without pressure (NoHP: kept on ice), at mHP (150 MPa, 37°C, 5 min), or at vHP [550 MPa, 60°C, 2.5 min (or 9 min for “vHP 9 min*”)] with or without 100 mM L-valine, TSB, or 2,500 IU/mL nisin at initial dormant spore concentrations (N₀) of 10⁹ CFU/mL (B. subtilis) or 10⁸ CFU/mL (B. amyloliquefaciens). “mHP vHP” means an mHP treatment was immediately followed by a water batch switch of ~30 s for a subsequent vHP treatment. “37°C” means that samples were incubated at 37°C for 6 h for nutrient germination before a second vHP treatment. “80°C” means heat activation for improved nutrient germination at 80°C for 10 min. Error bars present standard deviations of independent experiments (n ≥ 3). ^* Data from spore batch 2 (N₀ of 10¹⁰ CFU/mL, plated without washing after heat treatment). ^{- - -}Detection limit. ^aEstimated values (N < 100 CFU/mL). Treatments of B. subtilis at mHP or vHP with nisin and L-alanine instead of L-valine are not shown but were similar (File S1: Table S1). Nis: nisin; Val: L-valine

Fig 4

Flow cytometric quantification of ungerminated B. subtilis spores in mHP-treated or dormant spore (NoHP) samples after washing and heat treatment (80°C, 20 min). Spores were treated in buffer (pH 7.0) at 150 MPa and 37°C for 5 min (mHP) with or without 100 mM L-valine or 2,500 IU/mL nisin. In regions of certain fluorescence signal heights (-H), the following spore subpopulations appear: PI- and SYTO16-negative, ungerminated spores in region “R1”; SYTO16-positive, germinated spores with an intact inner membrane in region “R2”; and PI-positive, germinated, and presumably inactivated spores with membrane damage in region “R3.” The samples were exactly the same as those used for plate count analysis shown in Fig. 3. Percentages of events per region are shown as the mean ± standard deviation of three experimental replicates. Val: L-valine; Nis: nisin.

Fig 5

Influence of heat treatment (80°C, 20 min) on the reduction of culturable dormant B. subtilis spores (N) in nisin-containing samples, which were washed and plated before or after heat treatment (80°C, 20 min). Dormant spores were treated without pressure (NoHP: kept on ice), at mHP of 150 MPa and 37°C for 5 min, or at vHP of 550 MPa and 60°C for 2.5 min, with 100 mM L-Val in buffer (pH 7.0) or with TSB, and 2,500 IU/mL Nis at an initial dormant spore concentration (N₀) of 10⁹ CFU/mL. “80°C” and “37°C” mean that samples were heat-activated at 80°C for 10 min and incubated at 37°C for 6 h for nutrient germination before a second vHP treatment. Error bars present standard deviations of independent experiments (n ≥ 3). ^{- - -}Detection limit. Val: L-valine; Nis: nisin.

Fig 6

Influence of sample dilution or washing after heat treatment on the reduction of culturable dormant B. subtilis spores. Dormant spores were treated in buffer (pH 7.0) without pressure (NoHP: kept on ice) or at vHP of 550 MPa and 60°C for 2.5 min, with or without 100 mM L-valine and 2,500 IU/mL nisin at an initial dormant spore concentration (N₀) of 10⁹ CFU/mL. Samples were (i) washed 3× before the heat treatment (80°C, 20 min) by three cycles of centrifugation and supernatant exchange with ACES buffer at pH 7.0 (standard procedure), (ii) washed 3× before and 1× after heat treatment (“washed after heat”), or (iii) not washed at all (“no wash”). For the calculation of the culturable ungerminated spore concentration (N), the undiluted or 10× (1:10) diluted vHP samples were plated on agar. For NoHP samples with nisin, N was similar for the plated dilutions, resulting in similar log₁₀ reductions (e.g., for NoHP with nisin and 100 mM L-alanine after heat treatment and standard washing procedure: 1:10⁵ dilution: −1.1 ± 0.1 log₁₀ units; 1:10⁶ dilution: −1.1 ± 0.1 log₁₀ units). Error bars present standard deviations of independent experiments (n ≥ 3). ^{- - -}Detection limit. Val: L-valine; Nis: nisin.

Fig 7

Summary of factors influencing the reduction of culturable ungerminated spores. The factors were combined with non-high-pressure-treated B. subtilis spores (NoHP) or with mHP (150 MPa, 37°C, 5 min) or vHP (550 MPa, 60°C, 2.5 min) treatments. The factor “37°C + 2. vHP” corresponds to a vHP treatment followed by incubation at 37°C and a second vHP treatment in the presence of nutrients and nisin. Refer to Fig. 1 for an overview of tested parameters. Refer to the Results and Discussion to read whether a factor promoting reduction reduced the number of culturable ungerminated spores by promoting HP germination or inhibiting their growth.