Optimized Freeze-Drying of Probiotic Bacteria: Enhancing Viability, Metabolic Activity and Shelf-life Stability

Abstract

Lactic acid bacteria (LAB), commonly used as probiotics, offer significant health benefits and are widely applied in the food and pharmaceutical industries. However, their sensitivity to environmental stressors poses a major challenge to maintaining viability and functionality during processing and storage. Freeze-drying is a widely adopted preservation method that helps maintain LAB's stability and effectiveness during manufacturing and storage. This study aims to develop an optimized protocol for freeze-drying LAB strains, covering the entire process from cultivation to ideal storage conditions. Commercially viable, probiotic microbes were freeze-dried, than analyzed by periodic viability and lactate dehydrogenase (LDH) activity assessments to evaluate the shelf-life and metabolic activity of the strains in storage. As a control, commercially available probiotic capsules were initially tested, which showed low viability and poor lactic acid (LA) production. In contrast, probiotic bacteria freeze-dried using the optimized protocol maintained high viability and demonstrated lactic acid production upon rehydration and incubation in liquid media.

Introduction

Lactic acid bacteria (LAB) are a diverse group of non-motile, gram-positive, acidtolerant, and non-spore forming coccobacillus bacteria that play an essential role in both natural and industrial fermentation processes. These bacteria have mechanisms to metabolize carbohydrates such as lactose, glucose, and sucrose as carbon sources to generate energy, with lactic acid as a byproduct (Reddy et al., 2008). LAB are widely used by the food and pharmaceutical industries due to their wide range of applications and beneficial properties. When consumed in adequate amounts, LAB confer various health benefits, including modulation of the gut microbiota, enhancement of the immune system, alleviation of gastrointestinal disorders such as celiac disease, and even potential anticarcinogenic effects (Kumari et al., 2011). However, LAB are highly sensitive to environmental stressors, including pH changes, nutrient availability, oxidative stress, and interactions with metabolites from competing bacteria that might affect their viability. This sensitivity poses challenges in the applications of large-scale production of LAB as probiotics. Optimizing the processing conditions for LAB strains is important to guaranteeing their survivability during manufacturing and storage, ensuring adequate concentrations for their various food and clinical applications (Sionek et al., 2024).

To address these challenges, preserving LAB viability through optimized processing methods is essential. Lyophilization, or freeze-drying, is a popular technique to manufacture shelf stable probiotic cultures. The process involves three key steps: freezing the microbial culture, sublimating ice under vacuum during primary drying, and removing residual moisture during secondary drying. The use of a lyoprotectant reagent allows long-term preservation while minimizing cellular damage (Fonseca et al., 2015). While each bacterial strain is unique, the selection of ingredients, nutritional matrices, and manufacturing processes is crucial to ensuring cell viability and probiotic stability, imperative for its effectiveness (Forssten et al., 2011).

This study aims to develop an optimized freeze-drying protocol to enhance the shelf-life stability and viability of LAB, ultimately improving performance and consistency compared to existing commercial products. Furthermore, to assess LAB functionality post-lyophilization, lactic acid production was quantified using a LDH assay.

Materials and Methods

Preparation of Starter Cultures: Nine strains of commercially available encapsulated lactic acid bacteria were purchased (Table 1). Each probiotic capsule was dissolved in 0.1% peptone water to prepare a suspension, and used to inoculate agar plates. The bacteria were streaked onto MRS (de Man, Rogosa, and Sharpe, BD Difco™) agar plates and incubated at 37°C for up to 72 hours for colony formation. Colonies were inoculated into liquid MRS media and incubated at 37°C without shaking for 24 hours.

Table 1: List of the Lactic acid bacterial strains that were analyzed.

Lactiplantibacillus plantarum	Lactobacillus crispatus
Ligilactobacillus salivarius	Lactobacillus gasseri
Limosilactobacillus reuteri	Bifidobacterium longum
Lactobacillus acidophilus	Lactococcus lactis
Lacticaseibacillus rhamnosus

Commercial Product Viability Testing: The viabilities of the commercially available bacteria were assessed by a serial dilution to extinction protocol (OPS Diagnostics). Commercial capsules were opened and suspended in 10 ml of sterile 0.1% peptone water. A 96 well dilution plate was prepared by dispensing 180 µl MRS broth into each well, followed by the addition, in triplicate, of 20 µl suspended bacteria into column 1 of the plate. The bacteria were mixed by re-pipetting and then aseptically serially diluted 1:10 down to row 11. Row 12 remained a negative control. The 96 well plate was covered and incubated at 37°C for 48 hours and then scored for viability. Wells that contained growth (appearing cloudy) were scored positive. The last column that displayed growth was, on average, calculated as the number of viable cells. Thus, if the last column was 7, then the pill was estimated to contain 1 x 10⁷ viable cells. This same method was used to measure viable cells in the freeze-dried cultures.

LAB Strain Verification by 16S Sequencing: LAB were cultured overnight at 37°C in preparation of long read 16S rDNA sequencing. The DNA was isolated using Synergy™ 2.0 DNA Extraction Kit (OPS Diagnostics). The V1-V9 region was amplified using primers and a PCR protocol previously described by Matsuo et al. (2021).

The forward primer (5’-TTTCTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTCAG-3’) and reverse primer (5’-ACTTGCCTGTCGCTCTATCTTCCGGYTACCTTGTTACGACTT-3’) were used for amplification. The PCR reaction was performed on a QuantStudio 5 Real-Time PCR Instrument (Applied Biosystems, ThermoFisher Scientific). PCR conditions consisted of an initial denaturation at 95°C for 3 minutes, followed by 5 cycles of 95°C for 15 seconds, 55°C for 15 seconds, and 72°C for 30 seconds, then, 30 additional cycles of 95°C for 15 seconds, 62°C for 15 seconds, and 72°C for 30 seconds, with a final extension step at 72°C for 1 minute. Linear/PCR sequencing was performed by Plasmidsaurus (San Francisco, CA) using Oxford Nanopore Technology with custom analysis and annotation. The obtained FASTA sequences were analyzed using the BLAST algorithm provided by the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990).

Culturing Conditions for Freeze Drying: All strains were cultured at 37°C in MRS medium 24 hours prior to freeze-drying. Once cultured, cells were pelleted by centrifugation at 8,000 x g for 5 minutes. The supernatant was decanted and the cells were resuspended in 7/10 of their original volume in Microbial Freeze Drying Buffer (OPS Diagnostics). This cell suspension was used for freeze-drying and for 0 Day viability.

In a simple side study with L. plantarum, freeze-drying of the bacteria was performed after 24, 48, and 72 hours of growth at 37 °C. The viability of those cells was measured using the serial dilution method before and after lyophilization to assess the effect of extended culturing on freeze-drying.

Freeze-Drying: The suspended strains were dispensed into 5 ml clear serum vials, in 500 µl aliquots. Split lyophilization stoppers (bungs) were inserted into each vial. A VirTis Advantage Plus shelf freeze dryer was used for processing, with an initial freezing at -40°C for 2 hours. Primary drying was performed at a pressure below 200 mTorr, with the shelf temperature set to -15°C for 16 hours. Secondary drying was done by raising the shelf to 20°C and held for 2 hours. The vials were stoppered under vacuum and then allowed to equilibrate at room temperature for 1 hour before viability testing. The remaining vials were crimp sealed with 13 mm Aluminum Tear-Off Seals. Viability testing was performed for all strains before freeze-drying as described below.

Storage and Use: The vials were removed from the freeze dryer and stored at 4°C until they were used. To activate the cultures, bacteria were aseptically reconstituted in 500 µl sterile water.

Viability/Stability Testing: The viability of the bacteria was assessed by serial diluting in a 96-well plate as described above (OPS Diagnostics). Cell viability was measured after freeze-dried cells were rehydrated with 500 µl sterile distilled water. Viability testing was performed for all strains before freeze-drying (Day 0), after freeze-drying (Day 1), and at intervals of 1, 2, 4, 8, 12, 16, 20, and 24 weeks in a 96-well plate.

Lactic Acid Testing: Lactic acid production was compared between the commercial products and the freeze dried bacteria after 16 weeks of storage at 4°C. For the assay, commercial capsules and freeze-dried vials were suspended in 500 µl of ddH2O, and 200 µl of the suspended sample was inoculated into 20 ml of MRS broth. The culture was incubated for 6 hours at 37°C. The culture was centrifuged, and the supernatant was transferred to a separate tube. The supernatant was incubated at 65°C for 10 minutes to denature any lactate dehydrogenase present in the supernatant to limit loss of lactic acid prior to the assay (OPS Diagnostics, LLC). Lactic acid production was measured by a LDH assay (OPS Diagnostics). The assay uses a formazan dye and lactate dehydrogenase to detect lactic acid production from freeze-dried cultures rehydrated after storage.

The assay measures lactic acid concentration through a coupled reaction where lactate dehydrogenase (LDH) catalyzes lactate oxidation, reducing NAD+ to NADH. NADH then transfers electrons via phenazine methosulfate (PMS) to 2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), forming a red formazan product measurable at 490 nm. Lactic acid concentrations were determined using Beer’s Law (A = ebc), where A represents the absorbance measured at 490 nm for each sample, e is the molar absorptivity coefficient for INT (18,400 M^-1cm^-1), b is the optical path length (0.5 cm), and c corresponds to the concentration of the reduced sample, which is directly proportional to the lactic acid concentration in the assay.

Results and Discussion

Initial evaluation of viable cells in commercial probiotics was significantly different than levels stated on product packaging. The observed colony-forming unit (CFU) counts, given in Table 2, were noticeably lower than the values advertised on the product label. In the best cases, there was a three-log (10³) drop in viability. The poorest viability was observed with L. crispatus and L. acidophilus where the drop in viability was over seven logs (10⁷). As initial viability data on the cultures is unavailable, it is not known whether drop in viability was a consequence of poor culturing, inadequate freeze-drying, or from inherent characteristic of the LAB strain. In all situations, the viability was poor and estimated CFUs inaccurate.

Table 2: Actual versus advertised levels of viable probiotic bacteria in commercial preparations.

LAB species	Advertised CFUs	Actual CFUs	% Viability
L. plantarum	2 x 1010	1 x 104	0.0001
L. salivarius	1.5 x 109	1 x 105	0.0067
L. gasseri	1.5 x 109	1 x 106	0.067
L. crispatus	1.5 x 109	1 x 102	6.7 x 10-6
L. reuteri	1.5 x 109	1 x 106	0.067
L. acidophilus	1.5 x 109	1 x 102	6.7 x 10-6
L. rhamnosus	1.5 x 109	1 x 106	0.067
L. lactis	NA	1 x 107	NA
B. longum	2 x 109	1 x 105	0.005

LAB can be extremely sensitive to the different stressors they experience during freeze-drying, including osmotic pressure, temperature shifts, and local dehydration. These conditions can disrupt the cell membrane and cause cell death, observed in the loss of viability (Jalali et al., 2012). When the commercially available products were analyzed, it was determined they do not contain the CFU concentration advertised on the packaging, however it is unknown whether the viability decreased over time or whether it was low when packaged, but stable. In either case, ensuring cells retain high viability is essential for delivering their intended therapeutic benefits (Sarkar, 2018).

Sequencing the long-read 16S PCR product verified that identities of eight of the nine commercial strains were as claimed. The exception was Lactobacillus crispatus, based on repeated NCBI Blast searches, aligned primarily with Lacticaseibacillus rhamnosus. No further analysis, such as biochemical properties, was performed on these strains.

Each bacterial strain exhibits distinct behavior and varies in its growth rate. In our lab, it is common to freeze-dry bacteria when they are on their log phase of growth. However, high values of optical density may not correlate to viable cells or suitable physiological state for freeze-drying. The L. plantarum samples incubated for 48 and 72 hours exhibited a viability decrease of over 35% (Table 3) compared to 24 hour cultures. This demonstrates that higher cell densities are not necessarily optimal for good viability.

Table 3: Viability results from before and after freeze-drying of L. plantarum with different growth periods.

LAB species	Growth period before freeze drying	Day 0 (Log Dilution)	Day 1 (Log Dilution)
L. plantarum	24 hours	8	8
L. plantarum	48 hours	5	5
L. plantarum	72 hours	4	1

The primary objective of probiotic LAB is to produce organic acids, e.g., lactate. Thus, the effectiveness of commercial preparations versus freeze-dried cultures was evaluated for lactic acid production after a short culturing period. It is important to consider that these cultures are the same organisms but processed differently. After inoculating both the commercial and freeze-dried cultures into MRS broth, followed by a six-hour incubation, culture broth was harvested, cleared and measured for lactic acid.

Lactic acid was detected in all samples after incubation, however the LAB strains freeze-dried in-house produced significantly higher concentrations of lactic acid. Generally, the in-house freeze-dried LAB preparations had lactic acid concentrations 15 times higher than commercial probiotic capsules (Figure 1). As probiotics are routinely taken daily, a culture with faster acid production would plausibly be more desirable. To test the stability of the in-house cultures, the viability analysis was performed before freeze-drying, immediately following freeze-drying, and at 1, 2, 4, 8, 12, 16, 20, and 24 weeks post freeze-drying. The LAB viability remained relatively high over the period tested (Figure 2), which demonstrates that the ability of cultures to produce lactic acid was associated with viability of the strains.

Figure 1: Comparison of lactic acid concentrations in freeze-dried vials between samples from commercial preparations and OPS Diagnostics.

Figure 2: Viability of LAB species, stored at 4°C, assessed using a 96-well plate at regular intervals.

Conclusions

Lactic acid bacteria (LAB) play a critical role in promoting gut health through the production of antimicrobial metabolites and enhancement of the host immune response, while also aiding in carbohydrate digestion, particularly lactose (Sarkar, 2018; Kumari et al., 2011). The efficacy of probiotic products, however, is closely tied to the viability and metabolic activity of the strains they contain (Forssten et al., 2011; Sionek et al., 2024). Our findings revealed that several commercial probiotic formulations exhibited significantly reduced LAB viability and lactic acid productivity after culturing, raising concerns about their functional effectiveness.

In contrast, freeze-drying LAB in Microbial Freeze Drying Buffer (OPS Diagnostics) and storing them at 4°C resulted in markedly higher cell viability and sustained metabolic activity, specifically lactic acid production, aligning with previous findings on the critical role of optimized preservation protocols (Fonseca et al., 2015; Jalali et al., 2012). These results underscore the importance of carefully controlled freezedrying and storage conditions to maintain the functional integrity of probiotic formulations. Although the current study was conducted under laboratory conditions, ongoing research aims to replicate these protocols at an industrial scale, thereby bridging the gap between laboratory optimization and commercial viability (Reddy et al., 2008; Matsuo et al., 2021). Collectively, this work supports the need for improved manufacturing and preservation strategies to enhance the quality and reliability of probiotic products.

References

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

Fonseca, F., Cenard, S., & Passot, S. (2015). Freeze-drying of lactic acid bacteria. Methods in molecular biology (Clifton, N.J.), 1257, 477–488. https://doi.org/10.1007/978-1-4939-2193-5_24

Forssten, S. D., Sindelar, C. W., & Ouwehand, A. C. (2011). Probiotics from an industrial perspective. Anaerobe, 17(6), 410–413. https://doi.org/10.1016/j.anaerobe.2011.04.014

Jalali, M., Abedi, D., Varshosaz, J., Najjarzadeh, M., Mirlohi, M., & Tavakoli, N. (2012). Stability evaluation of freeze-dried Lactobacillus paracasei subsp. tolerance and Lactobacillus delbrueckii subsp. bulgaricus in oral capsules. Research in pharmaceutical sciences, 7(1), 31–36.

Kumari, A., Catanzaro, R., & Marotta, F. (2011). Clinical importance of lactic acid bacteria: a short review. Acta bio-medica: Atenei Parmensis, 82(3), 177–180.

Lactate Dehydrogenase Protocol. OPS Diagnostics, https://opsdiagnostics.com/applications/applicationtable/lactatedehydrogenaseprotocol.htm

Lyophilization Reagents Shelf-Life Study. OPS Diagnostics, https://opsdiagnostics.com/notes/lyoshelflife.htm.

Matsuo, Y., Komiya, S., Yasumizu, Y. et al. (2021). Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution. BMC Microbiol 21, 35. https://doi.org/10.1186/s12866-021-02094-5

Reddy, G., Altaf, Md., Naveena, B. J., Venkateshwar, M., & Kumar, E. V. (2008). Amylolytic bacterial lactic acid fermentation — a review. Biotechnology Advances, 26(1), 22–34. https://doi.org/10.1016/j.biotechadv.2007.07.004

Sarkar, S. (2018), "Whether viable and dead probiotic are equally efficacious?", Nutrition & Food Science, Vol. 48 No. 2, pp. 285300. https://doi.org/10.1108/NFS-07-2017-0151

Sionek, B., Szydlowska, A., Trzaskowska, M., & Kolozyn-Krajewska, D. (2024, June 4). The impact of physicochemical conditions on lactic acid bacteria survival in food products. MDPI. https://doi.org/10.3390/fermentation10060298