Enhancing the Survival Rate and Population Growth of Heyndrickxia Coagulans Spores for Use in Functional Foods

Abstract

Background and Objective: The delivery of probiotics using functional foods or supplements (e.g., capsules, tablets, or sachets) as carriers is an important strategy to provide health benefits, as it is necessary to maintain 10⁶-10⁹ colony-forming units (CFU) per gram of the probiotic strain alive and active at the time of consumption for effectiveness. This study investigated the effects of batch and fed-batch fermentation methods on increasing the cell and spore populations of Heyndrickxia coagulans MTCC 5856 (formerly Bacillus coagulans MTCC 5856). Material and Methods: Batch and fed-batch fermentation strategies were applied to evaluate their effects on vegetative growth and sporulation. The protective effects of various cryoprotectants (sorbitol, sucrose, inulin, calcium lactate, manganese chloride, and skim milk) were assessed during freeze-drying. Spore resistance under simulated gastrointestinal conditions and stability in two functional food matrices (pastille and coffee mix) were also evaluated. Results and Conclusion: Skim milk increased sporulation from 63% to 88%, with >80% spore viability in GI conditions and 85–98% stability in food matrices after 6 months. This study provides the first comparative analysis of batch versus fed-batch fermentation on H. coagulans MTCC 5856 sporulation in industrial food matrices, revealing glucose’s inhibitory effect and skim milk’s superior cryoprotective efficacy. These findings highlight the importance of optimizing fermentation conditions and applying suitable cryoprotectants to enhance the long-term viability and application of H. coagulans in functional food formulations. Keywords: Batch fermentation, Freeze-drying, Functional foods, Gastrointestinal resistance, Heyndrickxia coagulans, Probiotics, Sporulation, Spore stability Introduction Efforts to improve nutrition to promote health and prevent chronic diseases have led to the exploration of various food components and the production of functional foods [1]. Probiotics are live microorganisms that positively impact the host's health by balancing the natural microbes in the intestine, provided they are consumed in adequate amounts. Common probiotics, such as those from the Lactobacillus and Bifidobacterium genera, exhibit effective probiotic activity; however, their survival rates are generally low, making them quite vulnerable. Conditions of fermentation, freezing, thawing, drying, and additives for cell protection are among the factors that affect the survival of these microorganisms during the production of probiotic food [2]. Also, the digestive conditions of the body and stressful factors can cause the loss of a significant number of probiotic cells in the digestive system [3].  Bacillus spores are highly resilient, allowing them to endure the demanding conditions of product production and storage, as well as the challenges posed by digestive system, such as exposure to stomach acid and bile [4). Among the numerous Bacillus species, only a select few strains are available for use as human probiotic. One such strain is H. coagulans, which has recently garnered attention from researchers and food manufacturers due to its unique characteristics that combine traits of both the Bacillus and Lactobacillus genera [3]. This non-GMO probiotic is generally recognized as safe (GRAS), can tolerate high temperatures, and exhibits genetic stability over several years of commercial production. H. coagulans can survive for decades in harsh environments without cell division by forming spores [5]. In recent research, its clinical effectiveness has been confirmed to control hypercholesterolemia, lactose intolerance, digestive disorders, and diarrhea [6, 7]. Compared to other probiotic bacterial strains, the spore nature of H. coagulans has ensured its stability and vitality in functional foods and has not shown an adverse effect on the sensory and nutritional characteristics of the products [6, 8]. Table 1 presents a summary of the limited research regarding how various factors, such as the type of drying method and the application of protectants during the cell drying process, affect cell viability, sporulation, and the persistence of bacteria under simulated conditions of the digestive system, as well as during production and storage process. Despite the growing interest in H. coagulans, several gaps remain in the current literature. Comparative studies investigating the influence of batch versus fed-batch fermentation on its vegetative growth and sporulation are limited. Additionally, systematic evaluations of various cryoprotectants in preserving spore viability during freeze-drying are lacking. Furthermore, few studies have explored its resilience under simulated gastrointestinal conditions or assessed its long-term viability in real food matrices such as pastille and coffee mix under ambient storage. Pastille and coffee mix were selected in this study based on a direct request from Master Foodeh Food Industries (Tehran, Iran), which aims to develop probiotic-enriched versions of their commercial products. These food matrices represent two distinct formulation challenges, one being a gelatin-based, thermally processed confectionery product and the other a dry beverage powder intended for reconstitution with boiling water. Both products were evaluated under factory-scale conditions, confirming their feasibility for industrial application. The aim of this study was to evaluate the impact of fermentation strategy and cryoprotectants on the viability of H. coagulans, assess its resistance to simulated gastrointestinal conditions, and determine its shelf stability when incorporated into functional food products. Assessing the viability of H. coagulans in these real food systems aligns with the overall objective of this study and allows evaluation of its functional stability and potential use in commercially viable probiotic foods. Notably, this study provides the first comparative analysis of batch versus fed-batch fermentation on H. coagulans MTCC 5856 sporulation in industrial food matrices, showing that glucose inhibits sporulation and skim milk provides superior cryoprotective efficacy. Materials and Methods 2.1. Chemicals, solvents and other compounds Casein peptone, D(+)-Glucose, yeast extract, tryptone, agar, sodium chloride, sorbitol, sucrose, inulin, calcium lactate, magnesium sulfate heptahydrate (MgSO₄·7H₂O), manganese sulfate monohydrate (MnSO₄·H₂O), ferrous sulfate heptahydrate (FeSO₄·7H₂O), calcium carbonate (CaCO₃), sodium acetate (CH₃COONa), and skim milk were obtained from Merck (Darmstadt, Germany). Phosphate buffer was purchased from Bio Idea (Tehran, Iran). Pepsin and trypsin were acquired from Sigma-Aldrich (St. Louis, MO, USA), and bile oxalate was obtained from Ibresco (Iran). Industrial pastille and coffee mix were produced by Master Foodeh Food Industries Company (MFFIC, Tehran, Iran) using standard commercial procedures. 2.2. Microorganism and Culture Conditions Heyndrickxia coagulans MTCC 5856 was obtained from Maya Zist Farayand Company (Tehran, Iran). The strain was cultured in a modified medium containing (g/L): glucose 40, yeast extract 13.33, tryptone 13.33, MgSO₄·7H₂O 0.027, CaCO₃ 20, MnSO₄·H₂O 0.015, FeSO₄·7H₂O 0.024, NaCl 0.013, and sodium acetate (CH₃COONa) 0.67, based on formulation [15]. Stock cultures were preserved in 20% (v/v) glycerol at –80 °C. For inoculum preparation, a single colony was transferred into 10 mL of the same medium and incubated at 37 °C with shaking at 200 rpm for 16 h. This culture was then used to inoculate 100 mL of fresh medium at 5% (v/v), followed by incubation at 37 °C and 200 rpm for 24 h to obtain vegetative cells. For sporulation, the culture was subsequently incubated at 45 °C under static conditions for an additional 24 h. The selected temperature and incubation period were based on prior validation to balance sporulation efficiency and vegetative cell viability, considering both the nutrient composition and typical pH dynamics of medium. Although minor pH shifts occurred during this period, the primary driver for optimizing sporulation was the availability and balance of nutrients, which promoted maximal spore formation. The fermentation and sporulation conditions applied in this study were determined based on previously optimized parameters, as described in a separate manuscript currently under peer review. 2.3. Analysis Methods 2.3.1. Viable counts of H. coagulans The viable cell count of H. coagulans in each sample was determined by enumerating colony-forming units (CFU). Samples were serially diluted in sterile peptone water, and appropriate dilutions were plated using the pour plate method. Plates were incubated at 37 °C for 24 h, after which colonies were counted and expressed as CFU per gram of sample [16]. 2.3.2. H. coagulans Spore resistance To evaluate the thermal resistance of H. coagulans, the culture medium was subjected to heat treatment at 90 °C for 10 min and then rapidly cooled to 30 °C. This procedure was applied to eliminate vegetative cells while preserving heat-resistant spores. The treated samples were plated using the plate counting, and the plates were incubated at 37 °C for 48 h. Germinated spores formed visible colonies, which were counted and reported as CFU per gram [17]. 2.3.3. Statistical methodology All data were analyzed using Minitab software (version 21.4.2, Minitab LLC, USA), SPSS Statistics (version 26.0, IBM Corp., USA) and Microsoft Excel 2016 (Microsoft Corporation, USA). Results are presented as the mean ± standard deviation of triplicate measurements conducted in two independent experiments. Microbial counts were expressed as log₁₀ CFU/mL or log₁₀ CFU/g. Statistical significance was considered at P < 0.05. 2.4. Kinetics of bacterial growth, investigating pH changes and glucose consumption during bacterial growth To evaluate the growth kinetics of H. coagulans, the bacterial culture was incubated at 37 °C for 24 h. At 3-hour intervals, samples were taken to determine viable cell counts using the colony enumeration. Simultaneously, pH measurements of the culture medium were recorded using a digital pH meter. Glucose consumption was also monitored during the growth period. To quantify the residual glucose in the culture medium, a standard calibration curve was prepared using a glucose oxidase colorimetric kit (Pars Azmoon, Tehran, Iran) with concentrations of 0.1, 0.2, 0.4, 0.6, 1.0, 1.2, 1.4, 1.6, and 2.0 mg/mL, following the manufacturer’s protocol. Absorbance was measured at 540 nm using a spectrophotometer, and the glucose concentration in culture samples was calculated based on the standard curve at 3-hour intervals [18]. 2.5. Investigating the effect of feeding on cell growth and sporulation To investigate the effect of glucose supplementation on the growth and sporulation of H. coagulans, 250 µL of a 60% (w/v) glucose stock solution was added to 50 mL of culture medium at 3-hour intervals during incubation. Glucose feeding was performed based on the re-measured glucose concentration in the medium at each interval. The populations of vegetative cells and spores were determined at each sampling point using the pour plate method, as previously described. Glucose was chosen as the feeding substrate since it was already present in the base culture medium, and the aim was to examine how its continued availability might influence bacterial growth and sporulation dynamics. 2.6. Investigating the effect of different protectants on reducing damage to cells before transfer to freeze dryer The production of probiotic powder typically involves a freezing and drying process, during which dehydration can damage bacterial cell structures such as surface proteins, membranes, and cell walls. To mitigate these effects and enhance cell viability during freeze-drying, various protective agents were tested, including sorbitol, sucrose, inulin, calcium lactate, skim milk, and manganese chloride. Each protectant was added in equal concentration to identical volumes of bacterial suspension, and all samples were treated under the same conditions throughout cultivation and freeze-drying. Samples were initially frozen at –40 °C, then transferred to a freeze dryer and subjected to lyophilization for 18 h. The resulting powders were sealed in airtight polyethylene bags and stored at –4 °C to prevent moisture absorption. To assess the effectiveness of each protectant in preserving spore viability, pour plate culturing was performed on treated samples both before and after heat treatment (90 °C for 10 min). Plates were incubated at 37 °C for 48 h, and colony counts were compared between the different protectants and the control sample without any added protectant. Spore viability was assessed before and after freeze-drying by performing plate counting following heat treatment at 90 °C for 10 minutes to eliminate vegetative cells. Spore viability (%) was calculated by dividing the number of surviving spores after heat treatment by the total initial bacterial population (including vegetative cells and spores) before freeze-drying, and multiplying the result by 100. 2.7. Assessment of bacterial persistence under simulated gastrointestinal conditions  Probiotic microorganisms must survive the harsh conditions of the gastrointestinal tract to exert their beneficial effects. Therefore, the tolerance of H. coagulans to acidic pH, simulated gastric juice, and bile salts was evaluated under laboratory conditions. 2.7.1. Acid resistance: A fresh culture of H. coagulans was prepared, and 100 µL of microbial suspension was inoculated into 10 mL of culture medium adjusted to pH 2.5 and 4.0. The tubes were incubated at 37 °C, and after 3 h and 4 h, 1 mL of each sample was taken and analyzed using the colony enumeration. Viable cell counts were determined and expressed as CFU/mL. A minimum survival threshold of 10⁶ CFU/mL was considered acceptable [19, 20]. 2.7.2. Gastric juice resistance Simulated gastric juice was prepared using two separate media, each containing 2.3 g of pepsin and trypsin, and 2 g of sodium chloride per liter of distilled water, adjusted to pH 2.0. Control media were prepared with the same composition but adjusted to pH 7.0. Each medium was inoculated with 2% (v/v) of the H. coagulans suspension and incubated at 37 °C. Samples were taken at 0, 1, 2, 3, 4, and 24 h for viable cell count using the plate counting Survival above 10⁶ CFU/mL was considered indicative of resistance.[21] 2.7.3. Bile salt resistance  To assess the tolerance of H. coagulans spores to bile salts, two culture media were prepared containing 0.3% and 0.5% bile oxalate, along with a control medium without bile. Each tube was inoculated with 100 µL of microbial suspension and incubated at 37 °C. Viable cell counts were determined at 0 h and 8 h using the colony enumeration. The inhibition coefficient (Cinh) was calculated using equation .1 to quantify the inhibitory effect of bile salts on bacterial growth: Where T₀ and T₈ refer to the log CFU/mL values at time 0 and 8 hours, respectively. A Cinh value ≤ 0.4 indicates acceptable resistance to bile salts [12, 22]. 2.8. Evaluation of spore stability in food products The stability of H. coagulans spores was assessed in two functional food matrices: pastille and coffee mix, during both production and storage processes. 2.8.1. Pastille formulation  The base syrup was prepared by mixing glucose, sugar, and water at 100 °C. This mixture was combined with hydrated gelatin (prepared by dissolving gelatin in boiling water), food-grade coloring, citric acid, and essential oil. After cooling to an appropriate temperature, probiotic powder was added and the mixture was homogenized. The final mixture was poured into molds using a funnel and allowed to set for 24 h at room temperature. For microbiological analysis, randomly selected pastilles were dissolved in sterile peptone water and agitated in a shaker incubator for 1 h. Serial dilutions were then plated using the plate counting and incubated at 37 °C for 48 h. According to standard guidelines, a minimum viability of 10⁶ CFU per 100 g of pastille was considered acceptable. 2.8.2. Coffee mix formulation  Coffee mix powder was prepared from processed coffee beans using either spray drying or freeze drying. To simulate beverage preparation, a known quantity of probiotic powder was mixed with a single-serving coffee sachet (approximately 18 g), dissolved in 150 mL of boiling water, and stirred thoroughly.  To evaluate the stability of H. coagulans spores, samples were plated using the colony enumerationand incubated at 37 °C for 48 h. Colony counts were recorded and used to assess spore survival after exposure to product processing and heat. Results and Discussion 3.1. Kinetics of bacterial growth, pH variation, and glucose consumption To assess the growth behavior of H. coagulans, a batch fermentation was conducted, and key parameters including viable cell count, pH, and glucose concentration were monitored at 3-hour intervals for 24 h. Viable counts were determined by the plate counting, pH was measured using a digital pH meter, and residual glucose levels were quantified via spectrophotometric analysis. As shown in Figure 1, bacterial growth increased steadily until reaching its peak at 24 h, after which it declined, indicating entry into the death phase. These observations are consistent with the growth kinetics reported [23]. Glucose concentration in the medium decreased rapidly and was nearly depleted by 12 h, suggesting a high metabolic activity during the exponential phase. Following glucose exhaustion, the cells likely shifted to alternative nutrient sources. The pH of the culture medium showed a marked decline during the initial hours of growth, reaching its minimum around 18 h. This decrease corresponds to the accumulation of organic acids, primarily lactic acid, produced during active metabolism. After 18 h, pH levels began to rise slightly, which may be attributed to ammonium production from protein degradation or the consumption of lactate as a secondary substrate. After heat treatment, the final spore concentration was measured at 7.11 log CFU/mL, indicating successful sporulation under the tested conditions. 3.2. Investigation of the Effect of Feeding on Cell Growth and Sporulation In batch culture, due to the reduction of the initial concentration of food compounds in the culture medium and the lack of control of their concentration, formation and accumulation of the growth inhibitory compounds in the culture medium, bacterial growth quickly leaves the exponential growth phase and enters the stationary phase. Research has shown that employing a fed-batch culture method, which involves controlling nutrient concentrations, especially the limiting substrate, can significantly reduce the buildup of growth-inhibitory compounds. This strategy extends the exponential growth phase, ultimately improving the efficiency of the fermentation process. Therefore, in this research, to increase the number of vegetative cells and subsequently increase sporulation, the effect of the fed-batch process with pulse feeding was investigated on the growth of vegetative cells. Feeding was done according to the amount of glucose in the culture medium. According to the results illustrated in Figure 1, glucose was nearly completely consumed at 12 hours of fermentation. So, the first pulse feeding of glucose was initiated at this time. The glucose concentration in the culture medium was measured hourly, and approximately every three hours, when glucose was nearly depleted, an additional pulse of glucose was added to the culture. At that point, 250 μl of 60% glucose solution was added to 50 mL of the culture medium, after which the cell population was examined. Figure 2 shows the effect of feeding on pH changes and bacterial growth. A comparison of the results in Figures 1 and 2 indicates that although the bacterial growth rate has increased, the difference in vegetative cell populations between batch and fed-batch cultures when feeding at 12, 15, 18, 21 h of growth is not significant. This suggests that this kind of feeding had a slight effect on vegetative cell population growth. The pH continued to decline until hour of 24 and, in contrast to the batch process, the culture medium remained acidic throughout. At hour of 25 of cell growth, the culture medium was subjected to sporulation conditions for 24 h. After this period, pour plate cultures were performed both before and after heat treatment. However, the sporulation results in the fed-batch fermentation process were unsatisfactory; after 48 h, no spores were detected on the plates. While the total cell population before heat treatment was approximately 8.18 log CFU/ml, the vegetative cells did not convert to spores. A carbon source is essential for vegetative cell growth because it helps create an acidic environment. However, increasing its concentration has little effect on enhancing vegetative cell population and can inhibit spore production. This inhibition may be due to the negative effects of the carbon source on the synthesis of compounds necessary for spore formation. A study was conducted on the effect of glucose on the growth and sporulation of Bacillus subtilis. The results indicated that the population of vegetative cells increased with rising glucose concentrations, reaching a peak at 5g/l, after which it remained constant at higher glucose levels. However, the efficiency of spore formation decreased as glucose concentration increased. This is attributed to the fact that up to approximately 5g/l, all the glucose in the culture medium is used by the bacteria until the end of the logarithmic growth phase. Continue