Available online on 30.08.2026 at ijmspr.com

 International Journal of Medical Sciences and Pharma Research 

Open Access to Medical Science and Pharma Research

Copyright  © 2026 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited    

 

Factors Affecting the Stability of Probiotics in Pharmaceutical Products: A Comprehensive Review

Kohinur Begum ¹*, Shohana Dill Afrose ², Mahmuda Akter Setu ¹, Mehedi Hasan Shohan ¹

¹ Department of Pharmacy, School of Pharmaceutical Sciences, State University of Bangladesh, Dhaka-1461, Bangladesh

² Department of Pharmacy, ASA University Bangladesh, Dhaka-1207, Bangladesh

 

 

1. INTRODUCTION

Probiotic products are now established as a rapidly growing pharmaceutical and nutraceutical industry, having transitioned from an initiated nutraceutical niche product. Currently, probiotics are widely accepted in pharmaceutical and nutraceutical market as the focus has shifted to a mainstream focus. Based on an international consensus report by an expert panel organized by the International Scientific Association for Probiotics and Prebiotics, the official definition includes those microorganisms which upon ingestion by the host in sufficient quantities provide a beneficial health effect¹. It requires the organisms be alive, which puts viability as the core of efficacy, and the qualifying instructions with efficacy along adequate dose². Conventional small molecule drugs are robust when compared to probiotics, the active ingredients of which are life culture and they can subsequently lose activity from the time of manufacture³. Probiotics are used in various clinical settings due to their potential benefits, such as the prevention of antibiotic-associated diarrhea, treatment of irritable bowel syndrome, modulation of the gut microbiota and support for immune function^4,5. These benefits are achieved through a number of mechanisms, such as competitive exclusion of pathogens, generation of antimicrobial compounds, strengthening of the intestinal barrier, and modulation of the host immune system, all of which depend on the metabolic (living) activity of these cells^6,7. Formulation and preservation of fermented foods probiotic strains has further grown in interest due to their significant contribution to health^8,9. The health promoting effect of probiotics is not always the result of viability of cells. Commercial microbial probiotic products containing non-viable microorganisms, microbial cell components or microbial metabolites can also have beneficial biological activities^10,11. The most common genera used in the production of pharmaceutical products are relatively few. The dominant genera consist mainly of members of Lactobacillaceae, which have been reorganize into new genera in 2020 (Lacticaseibacillus, Lactiplantibacillus and Limosilactobacillus) along with Bifidobacterium species, the yeast Saccharomyces boulardii, and spore-forming bacteria such as Bacillus coagulans and Bacillus subtilis^12,13. These are all different organisms with different stability profiles, which the formulator must know and manage.

As science has grown faster in the commercial interest. Today, probiotics are billion-dollar internationalized products both functional foods and supplements and their market volume has been steadily increasing due to consumer demand for such preventive health foods and due to the growing recognition of the importance of the gut microbiome for human health¹⁴. This expansion is also called into question by tougher regulations. In some jurisdictions, probiotics for therapeutic or preventive action are considered to be live biotherapeutic products, which must comply with pharmaceutical manufacturing quality requirements including defined potency, purity and stability ^15,16. The label claim expressed per dose in colony-forming units (CFU) should continue to be the same until the expiration date³. However, the main challenges are the ability of a live probiotic to grow reduces over time, and the rate at which this occurs is dependent upon many interacting factors¹⁷. One of the most promising areas of probiotic formulation is the technological challenges associated with making a stable, high-count product, which have long been known as great constraints^18,19. All these challenges start from fermentation and harvesting, their selection influences the robustness of the biomass, and continue through the following operations such as formulation, manufacturing process, packaging and storage^20,21. Technological, functional, and safety properties must be taken into consideration together as it is certainly beneficial to include a strain that is clinically promising and is stable to develop into a reliable product²².  Historically, industry has been managing the stability by over-formulating with cells beyond the label claim, to cover for anticipated losses³. The research on probiotic stability has been initiated by empirical observations on retaining probiotic cell viability in fermented foods and was extended to mechanistic studies at cellular and molecular levels. Initial studies studied the effect of temperature and pH on survival, and more recent research has focused on the effect of conditions associated with oxidative stress, osmotic pressure, and metabolic dormancy on longevity. These characteristic factors and formulation matrices affect this process. This knowledge has led to the exploration of new delivery approaches such as microencapsulation, prebiotic-enriched matrices, and optimized packaging that maintain viabilities. In addition to viability, there are other concerns with inclusion of probiotics in pharmaceutical products in terms of functional integrity. Functional integrity is the capacity of the organism to perform its intended biological effects, such as their capacity to bind to the intestinal mucosa, and modulate host immune responses, including metabolic activity such as the production of the short-chain fatty acids¹⁷. This has led to the regulatory consideration of functional outcome along with the CFU-dependent viability evaluation, highlighting the need for adding microbiological, biochemical and omics-based analyses to the stability evaluation protocols. The growing recognition, strain specific and environmental factors have underlined the need to adopt a systems-level approach to understanding probiotic stability. The stress response pathways that protect a strain from acid, bile salts (bile acids), or oxidative stress are interacting in complex ways with the stressors of the formulation. Hence, a product's stability is not just a property of the product, but also a biological factor that relies on the context of its administration, host factors and formulation²³. Microbiological factors are intrinsic stress resistance of the strain, spore-forming capacity and the physiological state of the cells at the time of preservation²⁴. Formulation factors include moisture content, water activity of a product, the pH of the matrix, presence of oxygen during high temperatures, excipients and protective agents are considered in the formulation^25,26. Processing factors are the factors that cause sub-lethal and lethal injury to cells during the fermentation, harvesting, drying, granulation and compression process^27,28. Lastly the rate at which the product breaks down after leaving the factory depends on the environmental and storing conditions such as the packaging protective nature, temperature, humidity and illumination¹⁷. The aim of this review study is to explore the understand the effectiveness of probiotic microorganisms, different environmental conditions, and factors affect the stability during formulation and manufacturing processes, packaging and storage associated with probiotic dosage forms.  

2. METHODOLOGY 

This research study was carried out in the form of a systematic and extensive literature review. Scientific literature articles, reviews, and publications relevant to the research subject were searched on various e-databases like Google Scholar, PubMed Central, Scopus, Web of Science, and AI-assisted literature platforms. Searches were performed using combinations of keywords probiotics, pharmaceutical probiotics, probiotic stability, viability, formulation factors, packaging, storage conditions, live biotherapeutic products, pharmaceutical probiotic formulation manufacturing process. Following the initial search, article titles and abstracts were screened to assess relevance. Subsequently, full-text articles were evaluated in detail to determine their suitability for inclusion, and manual screening of reference lists from relevant and highly cited publications was performed to identify additional seminal studies. 

3. PROBIOTIC PHARMACEUTICAL DOSAGE FORMS

There are a number of solid and, in fewer instances, liquid dosage forms for delivering probiotic pharmaceutical products. Dry, semisolid or solid forms dominate since they can be subjected to low water activity during storage, which is the most effective method of maintaining viability during storage²⁵. Hard gelatin or hydroxypropyl methylcellulose capsules containing a powder or granulate, which have the benefits of being sealed, dosed and ready to be coated with enteric materials to prevent release to the gastric fluid containing cells²⁹. There are also tablets manufactured for use, but the compression step applies to them and they are subject to mechanical and thermal stresses which affect their viability³⁰. Sachets and stick packs for free-flowing powders are generally used for pediatric and geriatric medicines and orally disintegrating tablet (ODTs)³. The organisms used in these products vary significantly in their tolerance and the dosage forms depend on the selected strains. Vegetative cells of the Lactobacilli spp. and Bifidobacterium spp. are moisture, oxygen and heat sensitive and dried then encapsulated in protective formulation and stored at refrigerated temperatures ¹⁷. Intrinsically, the spore-forming bacteria, especially B. coagulans, are much more stable: the bacterial endospore is a dormant dehydrated form which is resistant to heating, desiccation, as well as oxidative damage and damage caused by tablet compression^12,31. The yeast Saccharomyces boulardii falls stronger than most lactic acid bacteria, but is sensitive to heat and moisture³². Traditionally, the potencies of these products are very high and marketed products state a range of between 1 and several hundred billion CFU/dose, and the number of viable organisms at the end of shelf life should exceed the labelled value to assure regulatory approval and to ensure that the required dose delivered for clinical trials is met^2,3. Probiotic manufacturing process, formulation, packaging and storage conditions are all interactive and considered in each step to achieve the desired product (Figure 1).

 

 

                 

Figure 1: A diagram of factors influencing the stability of pharmaceutical probiotic products.

 

 

4. MICROBIOLOGICAL DETERMINANTS OF STABILITY

From a biological point of view, the stability of a probiotic product is limited in a perfect way right from the start by the biology of the organism selected. Stress tolerance is inherent to the strain and dictates the maximum attainable stability even prior to deciding on formulating²⁴. The susceptibility to stress is not only genera and species dependent, but also strain dependent since it is associated with differences in membrane composition, stress-storing solutes and sensitivity of stress-defense genes³³. Selection of strains is the first and perhaps most important stability decision in product development and screening of prospective strains for processing and storage robustness is now deemed good practice³. The cells survivability is strongly affected by their physiological state at the time of preservation. Cells harvested during the stationary phase are adapted for stress conditions and in general have better drying and storage tolerance than those harvested from the exponential phase, presumably because of the expression of a spectrum of general stress-response proteins and accumulation of protective compounds during stationary phase³⁴. Stress adaptation or preconditioning is a technique that exposes cultures to controlled mild stress prior to preservation, and results in greatly enhanced cultures. Cross-protective responses, generated by short heat-shock, by controlled acid or osmotic stress experienced by cells prior to drying or storage, strengthen cells against adverse impacts of drying and storage^33,35. Some of the mechanisms involve the production of heat shock proteins, cold shock proteins, and synthesis of membrane fatty acid composition to maintain fluidity, and accumulation of trehalose and glycine betaine²⁸. A large number of interacting biological and process parameters will ultimately determine the quality of the freeze-dried culture through the process of stable culture preparation, and the relative importance of these variables must be determined for each organism^36,37. The most important microbiological factor causing stability is the ability of the organism to produce endospore. Spore-forming probiotics, mostly of the genera bacillus, come in a dormant state bound in a multi-layered coat and containing dipicolinic acid and small acid-soluble proteins¹². These properties offer the superior protection against heat, desiccation, oxygen, mechanical stress and gastric acid which enable preservation of spore-based products at ambient temperature for years, and allow their survival in tablet compression and even during cooking³¹. The intrinsic stability seems to be driving the rising commercialization around spore-forming probiotics, and the growing need for spores to germinate and to be able to provide a benefit continues to be an active research area³⁸. But the stability of vegetative organisms, contrary, must be obtained primarily through the process design and formulation.   That is because spore-producing Bacillus spp. is more resistant to environmental and processing stresses due to its ability to form endospores whereas vegetative lactic acid bacteria/bifidobacteria are relatively sensitive. Saccharomyces boulardii has a moderate stability. A summary of the probiotic microorganisms used in pharmaceutical dosages forms and their general stability characteristics are shown in Table 1.

 

 

Table 1:  Microorganisms used in pharmaceutical probiotic products and their general stability characteristics.

Genera of probiotics	Examples	Factors Affecting Stability	Dosage forms	Ref
Lactobacillus/ Lacticaseibacillus spp.	L. rhamnosus, L. plantarum, L. reuteri	Sensitive to moisture, oxygen, heat and compression; refrigeration preferred	Capsules, sachets, powders, ODTs	17, 25
Bifidobacterium spp.	B. longum, B. breve, B. animalis subsp. lactis	Highly oxygen and heat sensitive; requires protective formulation	Capsules, sachets, powders	17,25
Spore-forming Bacillus spp.	B. coagulans, B. subtilis	Highly resistant to heat, desiccation, oxidation and compression	Tablets, capsules, sachets	12, 30
Yeast	Saccharomyces boulardii	Intermediate stability; sensitive to heat and moisture	Capsules, sachets, powders	32

 

 

5. FORMULATION FACTORS

Principal intrinsic and environmental factors influencing probiotic stability, their mechanisms of action, impact on probiotic viability, and commonly employed mitigation strategies are crucial factors for product formulations. Water activity, residual moisture, oxygen exposure, oxidative stress, formulation pH, and excipient compatibility collectively determine the stability of probiotic pharmaceutical products during manufacturing are summarized in Table 2.

 

 

 

Table 2: Intrinsic and environmental factors affecting probiotic stability, their mechanisms of action, and mitigation strategies.

Factors	Mechanism	Impact	Mitigation	Ref
Water activity	Glass transition of the dried matrix, increasing molecular mobility	Reduced probiotic viability	Maintain Aw 0.10–0.25; optimized drying; moisture barriers	25,39,40
Residual moisture	Oxidation, Maillard reaction, residual metabolism	Cell death	Minimize moisture; protective packaging	25,28,41
Hygroscopic excipients	Increase moisture uptake	Indirect viability loss	Use low-hygroscopic excipients	26
Oxygen/ROS	Oxidative damage to lipids & proteins	Membrane damage	O₂ barriers, N₂ flush, scavengers, antioxidants	17,26,42,43
Formulation pH	Disrupts enzymes & membranes	Reduced viability	Buffers; optimize pH	24,28
Reducing sugars	Maillard reaction; incompatibility	Protein damage	Compatibility screening	27,28

 

 

 

5.1 Water Activity and Residual Moisture

Water activity is the most important factor, among all, affecting the shelf life of dried probiotics. Water availability is prime factor to chemical and biological reaction, and it sets the permeability of the dried material²⁵. As in many fields, empirical and mechanistic studies have consistently demonstrated that low water activity (0.1 to 0.25) is the optimum range for preservation of most organisms, and survival gradually and significantly declines when water activity is above that range^39,40. There are a number of mechanisms that act together to mediate these relationships. Under very low water activity, the dried matrix is in an extremely high viscosity amorphous glassy state, where molecular diffusion rates are very low thus preventing undesirable reactions⁴⁰. When water is absorbed, the glass transition decreases, and once the matrix reaches a lower temperature than the storage temperature, the glass transition temperature drops below the storage temperature causing the matrix to change to a rubbery state where the molecular mobility changes greatly and the rates of damaging reactions will be increased²⁵. Increased water activity will enhance lipid oxidation and also the reaction of reducing sugars with proteins, resulting in the production of Maillard reactions, which consequently will cause damage to the cell components²⁸. It also allows for persisting metabolic activity and for the transfer of reactive species to susceptible locations like membranes and DNA⁴¹. However, even though excipients may vary in hygroscopicity, it has been shown that the selection will have a great impact on the moisture the product will absorb during processing and storage, and if excipients are too hygroscopic, a good formulation may be jeopardized²⁶. Therefore, the main goal of probiotic formulation and packaging is to ensure low residual moisture content.

5.2 Oxygen and Oxidative Stress

In dried powders which have a diminished capacity for antioxidant defenses, oxidative stress is especially deleterious. The use of oxygen barrier packaging, flushing with nitrogen and additives such as antioxidants can greatly enhance stability⁴². The great majority of probiotic strains are microaerophilic or facultative anaerobic strains and oxygen exposures are injurious. Many probiotic microorganisms, such as bifidobacterial and oxygen-sensitive lactobacilli, are adversely affected by oxygen. Reactive oxygen species (ROS) form upon exposure to oxygen, and they can oxidize proteins and lipids in the membrane, resulting in the destruction of the membrane integrity and cell death⁴³. The organism also exhibits a difference in the presence of protective enzymes and many of the lactic acid bacteria do not have catalase, and processing and storage in aerobic environments means that lactic acid bacteria with limited oxidative-defense systems would be vulnerable to the enzyme⁴³. At higher water activity and higher temperatures, the effect of oxygen increases, demonstrating interdependency between factors affecting stability of the product²⁶. Therefore, the practical approach to the control of oxidative damage of products is mainly based on the exclusion of oxygen by means of low permeable packaging, addition of oxygen scavengers, modified atmosphere packaging or vacuum packaging, and addition of antioxidant excipients. 

5.3 Hydrogen Ion Concentration (pH) 

One of the most important factors affecting probiotic stability is the hydrogen ion concentration (pH) as it directly affects the integrity of the bacterial membranes, the activity of enzymes, protein conformation and the internal homeostasis. Additionally, the pH of the formulation matrix, and the chemical nature of excipients, has an impact on stability. Extremes of acidity damage cells, and lactic acid bacteria that produce acid during fermentation can self-acidify their environment unless buffered²⁴. Alkalinity also affects membrane bound enzyme systems and causes leakage of intracellular components. Most probiotic strains are less resistant to alkaline stress and become non-viable very quickly during storage periods than in mild acidity²⁸. 

 5.4 Excipients 

Excipients are not inert with regard to viability. The sugars that can be reduced (such as lactose and glucose) can start Maillard reactions with proteins in cell and matrix, causing browning and loss of viability, especially at the high moisture and high temperature²⁸. On the other hand, proper selection of excipients will maintain stability of the cells and protection agents. The role of each ingredient is required to screen as protective, harmless, and the of potential compatibilities with probiotics during development of probiotic formulation²⁷.

6. MANUFACTURING PROCESS FACTORS

The manufacturing of probiotic products is a highly sensitive process. Because probiotics are sensitive microorganisms, processing factors such as strain selection, fermentation parameters, drying methods, and encapsulation heavily dictate the final product's viability and stability. Comparison of different technologies used in manufacturing process for probiotic products is given in Table 3.

 

 

Table 3: Comparison of different technologies used in manufacturing process for probiotic products.

Methods	Principle	Advantages	Limitations	Applications	Ref
Freeze-drying (Lyophilization)	Freezing followed by vacuum sublimation	High cell survival; excellent product stability; suitable for heat-sensitive probiotics	High cost, long processing time, energy-intensive	High-value pharmaceutical probiotics	27,44
Spray-drying	Atomization into hot air with rapid drying	Continuous, scalable, economical	Thermal and dehydration stress reduce viability	Large-scale probiotic powder production	45–47
Fluidized-bed drying	Drying suspended particles in controlled airflow	Uniform drying; suitable for coated granules	Process optimization required	Granules and coated formulations	28
Vacuum drying	Moisture removal under reduced pressure	Lower thermal stress than conventional drying	Slower than spray-drying; limited industrial use	Heat-sensitive formulations	28
Spray-freeze-drying	Atomization into cryogenic medium followed by freeze-drying	Excellent viability with porous particles	High processing cost and complexity	Specialized pharmaceutical applications	28
Encapsulation	Physical entrapment of probiotic cells within a protective polymer or biopolymer matrix	Protects against drying, oxygen, moisture, gastric acid, bile salts, and mechanical stress; improves storage stability and controlled intestinal release	Increased production cost; optimization of wall material, particle size, encapsulation efficiency, and release profile required	Pharmaceutical probiotics, functional foods, targeted intestinal delivery	42,48–50

 

 

6.1 Fermentation and Harvesting

In the industrial production of probiotics fermentation and harvesting are identified as critical steps. These processes determine the viability, stability, functionality, and quality of probiotic microorganisms. Stability is a design choice that is made at an early stage in the creation of a product. The physiological robustness of the cells is dependent on the fermentation conditions, the growth media and harvested time³⁴. Shear and osmotic stresses are created in downstream processes (such as centrifugation and concentration) and the quality of the concentrate containing any support additives used prior to drying determines the cell's response to the dehydration⁴⁵.

6.2 Drying Technologies

The common processing stage that determines the quality of most probiotic products is the drying stage, as the most important factor to ensure the arrest of vitality during storage is to reduce the water content to a stable and low level²⁷. One of the most damaging stresses cells would encounter during drying is the destruction of the hydration shells of membranes and proteins, the increase of the solute concentration and its potential to cause fatal phase changes in the lipid bilayer of membranes which occur upon drying. Removing water and maintaining the cell structure⁵¹ is a challenge when drying probiotics. High potential pharmaceutical probiotics are most commonly freeze-dried or lyophilized. The effect of the process is a freezing of the suspension which separates water through the sublimation process under vacuum, without the application of high temperatures used in thermal drying²⁷. The main causes of damage are associated with the freezing process, when ice crystals and solute dry out and the cells are stressed by the high concentration of solutes and the resulting ice crystals, and the post freezing dehydration that loss water in the cells, which is essential to maintain membrane and protein structure⁴⁴. Overall, the freeze-drying method produces a very high survival rate and high-quality dried products but is energy-consuming, slow and expensive²⁷. The economical alternative is also available such as spraying the suspension into a stream of hot air that is able to evaporate the water from the droplets in seconds⁴⁶. The major disadvantage is the heat stress and very rapid drying phenomenon that significantly impair viability, especially for heat sensitive strains⁴⁵. Survival is highly reliant on outlet temperature, type of carrier and protective agents, and strain strength and optimization of these conditions yield survival rates close to the levels for more tolerant organisms^46,52. Spray drying of lactic acid starter cultures has therefore been a topic of ongoing research to get viable lactic acid powders at a low cost⁴⁷. In addition, other alternative and emerging methods are considered such as fluidized-bed drying, vacuum drying and spray-freeze-drying with different compromise of cost, throughput, and gentle²⁸.

6.3 Compression and Granulation

Compression result in mechanical and frictional heat stress in tablet products which may damage the cell membrane and decrease the viability³⁰. Loss of viability is a function of the pressure applied, the ability of the excipients responds to compression, and the organism's ability to withstand the compression, where spore-forming strains remain much more resistant to compression than vegetative cells¹². Granulation prior to compression is useful in protecting cells by encapsulating them within a matrix, besides increasing the powders flow characteristics, however, wet-granulation techniques lead to rehydration with moisture and heat, which needs to be controlled²⁷. Again, these considerations tend to favor spore formers or encapsulated cells with strong capsules for tablet dosage forms.

6.4 Encapsulation Technologies

The principle of microencapsulation is also the principle of protection, but moreover the cells are separated from the surrounding by a particle which is almost enclosing the cell⁴². The technological  knowledge is developing and numerous review papers are reported on materials, methods and viability^53–55. Cells can be encapsulated to shield from stresses of oxygen, moisture, gastric acid and mechanical stresses, and further modified to release the cell in a desired location such as the intestine^48,49. Different materials and methods can be applied. However, the alginates are extruded or emulsified and are allowed to gel, and occasionally a Chitosan layer and/or additional layers are added, all of which are the most studied hydrocolloid gels^50,56. Spray-drying and spray-chilling can be utilized for the production of encapsulated microparticles in a single continuous process and fluidized-bed coating can be used for the coating of dried granules with a protective and functional layer^42,57. Many comparative studies on the survival of microencapsulated cells after processing and storage have been mentioned with varying results according to various technique   and the material used ^58,59. The easiest method to improving survival during drying and storage is by supplementation of protective agents. These compounds are effective through multiple mechanisms such as the hydrogen bonding water normally forms to the surface of membranes and proteins, amorphous formation of a glassy matrix which immobilizes and protects cells, and compounds act as a scavenger for reactive species or buffer the local environment^40,60. Disaccharides are the most frequently-used as protectants. In particular, trehalose is very effective for its ability to form a high glass transition temperature, stable glass; is non-reducing and is a natural protectant in many desiccation-tolerant organisms, due to its nature, have no negative side effects⁶¹. Combined mechanisms of vitrification and water replacement have been found for the simultaneous protection of membranes and proteins during drying of intact bacteria with either trehalose or sucrose⁶². In addition to reducing the availability of oxygen, antioxidants (such as ascorbate) added to cells also play an important role in ensuring the storage stability of the freeze-dried cells at a specific water activity⁶³. Applications of other sugars, like sucrose or lactose, of skinned milk, of maltodextrin and of various polysaccharides and proteins are also made and often utilized as mixtures^45,51. It is also reported that the prebiotic carbohydrates and cryoprotectant mixes were more successful in surviving the freeze-drying and storage process⁶⁴. It is still necessary to perform strain dependent optimization to find the best protectant based on the strain, and systematic screening during development²⁷. Although use of encapsulation can greatly enhance the in-process (processing and storage) and intestinal (GI transit) survival of the product. An important number of parameters need to be optimized, such as the encapsulation efficiency of the encapsulated cell, particle size and mechanical and chemical properties of the wall material, encapsulated cell release behavior^42,49. Therefore, the encapsulation process also needs to be taken into account in an integrated stabilization program when other factors like moisture, oxygen, temperature and packing are considered^65,66.

7. PACKAGING AND STORAGE FACTORS

The viability of probiotics during shelf life is critical to design the formulations and optimize the packaging strategies. Once the product is manufactured, storage temperature is the most important factor affecting the rate of loss of viability of a probiotic product. However, the rate of cell death rises while temperature increases. Cold-chain supply is recommended for the sensitivity of sensitive strains and product labeling is also essential with storage instructions. The ability and commercial priority of producing thermostable strains and formulations that store at ambient temperatures is highly significant³. Spore-forming probiotics, vegetative cells with very low water activity and well encapsulated probiotics are increased the range of product stability in time and temperature^12,25.

The shelf life of probiotics is determined from stability studies with packaged product stored under defined conditions and the number of viable cells is measured over time. Especially the ultraviolet light is a source of inducing photo-oxidative damage and therefore opaque or light-protective packages are preferred for sensitive products. The principles of pharmaceutical stability testing outlined in the International Council for Harmonization (ICH) guidelines offer a framework, which was developed for chemically defined drugs, and which are needed to be adapted for live organisms³. The packaging has to serve several protective roles such as high barrier to moisture and oxygen, exclude light, must be robust during transportation. Aluminum foil blisters, glass bottles and high barrier plastic bottles, integrated desiccants, sachet desiccants, oxygen scavengers, and vacuum and modified atmosphere packaging are used for such purposes⁶⁷. The moisture uptake during storage is dependent on storage humidity and the viability is highly sensitive to water activity, therefore, moisture control during storage is crucial²⁵. Real time tests are needed to optimize desired storage temperature and shelf life. Studies at high temperature and their interpretation with kinetic models can be also validated for the development of the particular product⁴⁰. 

8. REGULATORY IMPORTANCE OF PROBIOTIC STABILITY

The regulatory classification of probiotics varies across countries and is primarily determined by their intended use, health claims, dosage form, and the regulatory policies established by the respective national authorities. Pharmaceutical products which are intended to prevent, treat or cure a disease, can be considered to pharmaceutical manufacturing and quality requirements, as opposed to food processing and quality requirements as is done for products labeled as dietary supplements or foods^15,16. There are multiple requirements, including strain identity and purity, the ability to validate the viability assay, formal stability studies and data justifying the label claim and date of validity^3,68. Product quality is important core component with good manufacturing practice, genomic technique for strain authentication as well as contamination control^2,69. In market products, where viable counts were present and active, stability problems are reported, as in many independent probiotics survey products^70,71. A vital requirement is the regulatory stability which is considered to be a critical quality attribute (CQA) as good therapeutic efficacy would rely on viable and functionally active microbial count during the shelf-life period of the product being manufactured, processed or treated⁴⁸. The emphasis of regulatory information from the ICH⁷² and the United States Food and Drug Administration (FDA) is to identify and control formulation and manufacturing parameters that affect the stability of a product. Therefore, it is necessary to apply scientific principles when composing probiotic products like these, such as those using a suitable packaging and storing process. The regulatory paradigm for live biotherapeutic products is also evolving and has raised expectations for stability that is proven, rather than empirical, and for move towards rational, mechanism-based design¹⁶.

9. STRATEGIES TO ENHANCE STABILITY

Based on the factors discussed above, a classic list of strategies for making probiotics more stable is presented as each effective only when used in combination. The first is a selection of a strong strain, usually a species that has been tested for resistance to the stresses of the proposed process and storage conditions, and a spore-forming species with intrinsic resistance^3,12. Cells must be cultured and harvested in a state that best promotes their ability to withstand stress³³. The desired product should be dried to the low water activity required the process which is adapted to the heat sensitivity of the strain and the use of suitable protective agents and, formulate microencapsulation to protect the cells^25,42. The packaging used for storing the product must be considered at highest priority. The stabilization of probiotic pharmaceuticals will likely be influenced by a few developments in the near future. Technological innovations such as improvements to spray drying and new gentle drying processes to achieve similar high freeze-drying survival with economy^46,73. New approaches to capsule design and construction, such as the layer-by-layer technique and the use of prebiotic or biopolymer layer(s), provide new levels of protection and release control^49,50. In addition, methods in which probiotics are co-encapsulated with bioactive or prebiotic ingredients to provide stability as well as added functionality⁷⁴. As the interest in spore forming probiotics and the next generation organisms like: Akkermansia muciniphila and Faecalibacterium prausnitzii is gathering momentum, it will bring novel stabilization challenges when compared to the established probiotics⁷⁵. Better verification of rapid validated viability testing, especially the flow-cytometric and molecular methods that are able to characterize the physiological state of cells, will improve the quality control in addition to the prediction of the shelf life^76,77.

10. CONCLUSIONS

 A number of factors could influence the viability including water activity, temperature, presence of oxygen, manufacturing process, packaging system and storage conditions. All these can cause cell damage by disrupting the cell membrane, denaturing proteins, causing oxidative damage and affecting cell death, leading to a decrease in probiotic survival. Strain specific parameters, cellular physiology, formulation ingredients as well as advanced drying technologies and microencapsulation are also important factors that contribute to the stability of probiotics. Therefore, there is a need for a comprehensive strategy that covers the selection of the strain, optimization of the process, formulation, packaging and storage conditions for probiotics to ensure their stability over time. However, the stability of probiotics in pharmaceutical products is a multifactorial phenomenon that can be traced back to the interaction between various intrinsic characteristics of the microbial agents, environmental factors, production process, and regulatory requirements. This review provides an in-depth overview of the major factors affecting probiotic stability and highlights current and emerging strategies to enhance the quality, shelf life of probiotic pharmaceutical products.

Acknowledgement: The authors would like to thank the institution for its support for this study.

Conflict of interest: The authors declared no conflict of interest.

Funding: No funding sources.

REFERENCES

1. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014 Aug 1;11(8):506-14. https://doi.org/10.1038/nrgastro.2014.66 PMid:24912386

2. Sanders ME, Merenstein DJ, Reid G, Gibson GR, Rastall RA. Probiotics and prebiotics in intestinal health and disease: from biology to the clinic. Nat Rev Gastroenterol Hepatol. 2019 Oct;16(10):605-16. https://doi.org/10.1038/s41575-019-0173-3 PMid:31296969 PMCid:PMC8416245

3. Fenster K, Freeburg B, Hollard C, Wong C, Rønhave Laursen R, Ouwehand AC. The Production and Delivery of Probiotics: A Review of a Practical Approach. Microorganisms. 2019 Mar 17;7(3):83. https://doi.org/10.3390/microorganisms7030083 PMid:30884906 PMCid:PMC6463069

4. McFarland LV. Efficacy of Single-Strain Probiotics Versus Multi-Strain Mixtures: Systematic Review of Strain and Disease Specificity. Dig Dis Sci. 2021 Mar;66(3):694-704. https://doi.org/10.1007/s10620-020-06244-z PMid:32274669

5. Suez J, Zmora N, Segal E, Elinav E. The pros, cons, and many unknowns of probiotics. Nat Med. 2019 May;25(5):716-29. https://doi.org/10.1038/s41591-019-0439-x PMid:31061539

6. Bron PA, Van Baarlen P, Kleerebezem M. Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa. Nat Rev Microbiol. 2012 Jan;10(1):66-78. https://doi.org/10.1038/nrmicro2690 PMid:22101918

7. Sánchez B, Delgado S, Blanco‐Míguez A, Lourenço A, Gueimonde M, Margolles A. Probiotics, gut microbiota, and their influence on host health and disease. Mol Nutr Food Res. 2017 Jan;61(1):1600240. https://doi.org/10.1002/mnfr.201600240 PMid:27500859

8. Marco ML, Sanders ME, Gänzle M, Arrieta MC, Cotter PD, De Vuyst L, et al. The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on fermented foods. Nat Rev Gastroenterol Hepatol. 2021 Mar;18(3):196-208. https://doi.org/10.1038/s41575-020-00390-5 PMid:33398112 PMCid:PMC7925329

9. Markowiak P, Śliżewska K. Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients. 2017 Sep 15;9(9):1021. https://doi.org/10.3390/nu9091021 PMid:28914794 PMCid:PMC5622781

10. Lahtinen SJ. Probiotic viability - does it matter? Microb Ecol Health Dis. 2012 Jun 18;23(0). https://doi.org/10.3402/mehd.v23i0.18567 PMid:23990833 PMCid:PMC3747757

11. Sanders ME. Probiotics: Definition, Sources, Selection, and Uses. Clin Infect Dis. 2008 Feb;46(s2):S58-61. https://doi.org/10.1086/523341 PMid:18181724

12. Cuentas AM, Deaton J, Khan S, Davidson J, Ardita C. The Effect of Bacillus subtilis DE111 on the Daily Bowel Movement Profile for People with Occasional Gastrointestinal Irregularity. J Probiotics Health. 2017;05(04). https://doi.org/10.4172/2329-8901.1000189

13. Zheng JS, Luan J, Sofianopoulou E, Sharp SJ, Day FR, Imamura F, et al. The association between circulating 25-hydroxyvitamin D metabolites and type 2 diabetes in European populations: A meta-analysis and Mendelian randomisation analysis. PLOS Med. 2020 Oct;17(10):1-21. https://doi.org/10.1371/journal.pmed.1003394 PMid:33064751 PMCid:PMC7567390

14. Reid G, Gadir AA, Dhir R. Probiotics: Reiterating What They Are and What They Are Not. Front Microbiol. 2019 Mar 12;10:424. https://doi.org/10.3389/fmicb.2019.00424 PMid:30930863 PMCid:PMC6425910

15. Dreher-Lesnick SM, Stibitz S, Carlson, Jr. PE. U.S. Regulatory Considerations for Development of Live Biotherapeutic Products as Drugs. Britton RA, Cani PD, editors. Microbiol Spectr. 2017 Sep 22;5(5):5.5.11. https://doi.org/10.1128/microbiolspec.BAD-0017-2017 PMid:28975881 PMCid:PMC11687541

16. Cordaillat-Simmons M, Rouanet A, Pot B. Live biotherapeutic products: the importance of a defined regulatory framework. Exp Mol Med. 2020 Sep;52(9):1397-406. https://doi.org/10.1038/s12276-020-0437-6 PMid:32908212 PMCid:PMC8080583

17. Tripathi MK, Giri SK. Probiotic functional foods: Survival of probiotics during processing and storage. J Funct Foods. 2014 Jul;9:225-41. https://doi.org/10.1016/j.jff.2014.04.030

18. Mattila-Sandholm T, Myllärinen P, Crittenden R, Mogensen G, Fondén R, Saarela M. Technological challenges for future probiotic foods. Int Dairy J. 2002 Jan;12(2-3):173-82. https://doi.org/10.1016/S0958-6946(01)00099-1

19. Ross RP, Desmond C, Fitzgerald GF, Stanton C. Overcoming the technological hurdles in the development of probiotic foods. J Appl Microbiol. 2005 Jun;98(6):1410-7. https://doi.org/10.1111/j.1365-2672.2005.02654.x PMid:15916653

20. Champagne CP, Gardner NJ, Roy D. Challenges in the Addition of Probiotic Cultures to Foods. Crit Rev Food Sci Nutr. 2005 Jan;45(1):61-84. https://doi.org/10.1080/10408690590900144 PMid:15730189

21. Lacroix C, Yildirim S. Fermentation technologies for the production of probiotics with high viability and functionality. Curr Opin Biotechnol. 2007 Apr;18(2):176-83. https://doi.org/10.1016/j.copbio.2007.02.002 PMid:17336510

22. Saarela M, Mogensen G, Fondén R, Mättö J, Mattila-Sandholm T. Probiotic bacteria: safety, functional and technological properties. J Biotechnol. 2000 Dec;84(3):197-215. https://doi.org/10.1016/S0168-1656(00)00375-8 PMid:11164262

23. Binda S, Hill C, Johansen E, Obis D, Pot B, Sanders ME, et al. Criteria to Qualify Microorganisms as "Probiotic" in Foods and Dietary Supplements. Front Microbiol. 2020;Volume 11-2020. https://doi.org/10.3389/fmicb.2020.01662 PMid:32793153 PMCid:PMC7394020

24. Corcoran B, Stanton C, Fitzgerald G, Ross R. Life Under Stress: The Probiotic Stress Response and How it may be Manipulated. Curr Pharm Des. 2008 May 1;14(14):1382-99. https://doi.org/10.2174/138161208784480225 PMid:18537661

25. Higl B, Kurtmann L, Carlsen CU, Ratjen J, Först P, Skibsted LH, et al. Impact of Water Activity, Temperature, and Physical State on the Storage Stability of Lactobacillus paracasei ssp. paracasei Freeze‐Dried in a Lactose Matrix. Biotechnol Prog. 2007 Jan;23(4):794-800. https://doi.org/10.1021/bp070089d PMid:17636886

26. Vesterlund S, Salminen K, Salminen S. Water activity in dry foods containing live probiotic bacteria should be carefully considered: A case study with Lactobacillus rhamnosus GG in flaxseed. Int J Food Microbiol. 2012 Jul;157(2):319-21. https://doi.org/10.1016/j.ijfoodmicro.2012.05.016 PMid:22663981

27. Broeckx G, Vandenheuvel D, Claes IJJ, Lebeer S, Kiekens F. Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics. Int J Pharm. 2016 May;505(1-2):303-18. https://doi.org/10.1016/j.ijpharm.2016.04.002 PMid:27050865

28. Santivarangkna C, Kulozik U, Foerst P. Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J Appl Microbiol. 2008 Jul;105(1):1-13. https://doi.org/10.1111/j.1365-2672.2008.03744.x PMid:18266696

29. Govender M, Choonara YE, Kumar P, Du Toit LC, Van Vuuren S, Pillay V. A Review of the Advancements in Probiotic Delivery: Conventional vs. Non-conventional Formulations for Intestinal Flora Supplementation. AAPS PharmSciTech. 2013 Sep 25;15(1):29-43. https://doi.org/10.1208/s12249-013-0027-1 PMid:24222267 PMCid:PMC3909163

30. Klayraung S, Viernstein H, Okonogi S. Development of tablets containing probiotics: Effects of formulation and processing parameters on bacterial viability. Int J Pharm. 2009 Mar 31;370(1-2):54-60. https://doi.org/10.1016/j.ijpharm.2008.11.004 PMid:19059323

31. Konuray G, Erginkaya Z. Potential Use of Bacillus coagulans in the Food Industry. Foods. 2018 Jun 13;7(6):92. https://doi.org/10.3390/foods7060092 PMid:29899254 PMCid:PMC6025323

32. Czerucka D, Piche T, Rampal P. Review article: yeast as probiotics - Saccharomyces boulardii. Aliment Pharmacol Ther. 2007 Sep;26(6):767-78. https://doi.org/10.1111/j.1365-2036.2007.03442.x PMid:17767461

33. Mills S, Stanton C, Fitzgerald GF, Ross Rp. Enhancing the stress responses of probiotics for a lifestyle from gut to product and back again. Microb Cell Factories. 2011 Dec;10(S1):S19. https://doi.org/10.1186/1475-2859-10-S1-S19 PMid:21995734 PMCid:PMC3231925

34. Van De Guchte M, Serror P, Chervaux C, Smokvina T, Ehrlich SD, Maguin E. Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek. 2002 Aug;82(1-4):187-216. https://doi.org/10.1023/A:1020631532202 PMid:12369188

35. Desmond C, Stanton C, Fitzgerald GF, Collins K, Paul Ross R. Environmental adaptation of probiotic lactobacilli towards improvement of performance during spray drying. Int Dairy J. 2002 Jan;12(2-3):183-90. https://doi.org/10.1016/S0958-6946(02)00040-7

36. Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P. Relevant factors for the preparation of freeze-dried lactic acid bacteria. Int Dairy J. 2004 Oct;14(10):835-47. https://doi.org/10.1016/j.idairyj.2004.02.001

37. Morgan CA, Herman N, White PA, Vesey G. Preservation of micro-organisms by drying; A review. J Microbiol Methods. 2006 Aug;66(2):183-93. https://doi.org/10.1016/j.mimet.2006.02.017 PMid:16632005

38. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives. Front Microbiol. 2017 Aug 10;8:1490. https://doi.org/10.3389/fmicb.2017.01490 PMid:28848511 PMCid:PMC5554123

39. Ananta E, Volkert M, Knorr D. Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG. Int Dairy J. 2005 Apr;15(4):399-409. https://doi.org/10.1016/j.idairyj.2004.08.004

40. Aschenbrenner M, Kulozik U, Foerst P. Evaluation of the relevance of the glassy state as stability criterion for freeze-dried bacteria by application of the Arrhenius and WLF model. Cryobiology. 2012 Dec;65(3):308-18. https://doi.org/10.1016/j.cryobiol.2012.08.005 PMid:22964396

41. Teixeira P, Castro H, Mohácsi‐Farkas C, Kirby R. Identification of sites of injury in Lactobacillus bulgaricus during heat stress. J Appl Microbiol. 1997 Jul;83(2):219-26. https://doi.org/10.1046/j.1365-2672.1997.00221.x PMid:9281825

42. Burgain J, Gaiani C, Linder M, Scher J. Encapsulation of probiotic living cells: From laboratory scale to industrial applications. J Food Eng. 2011 Jun;104(4):467-83. https://doi.org/10.1016/j.jfoodeng.2010.12.031

43. Talwalkar A, Kailasapathy K. A Review of Oxygen Toxicity in Probiotic Yogurts: Influence on the Survival of Probiotic Bacteria and Protective Techniques. Compr Rev Food Sci Food Saf. 2004 Jul;3(3):117-24. https://doi.org/10.1111/j.1541-4337.2004.tb00061.x PMid:33430563

44. Fonseca F, Cenard S, Passot S. Freeze-Drying of Lactic Acid Bacteria. In: Wolkers WF, Oldenhof H, editors. Cryopreservation and Freeze-Drying Protocols [Internet]. New York, NY: Springer New York; 2015 [cited 2026 Jun 23]. p. 477-88. (Methods in Molecular Biology). Available from: https://link.springer.com/10.1007/978-1-4939-2193-5_24 https://doi.org/10.1007/978-1-4939-2193-5_24 PMid:25428024

45. Fu N, Chen XD. Towards a maximal cell survival in convective thermal drying processes. Food Res Int. 2011 Jun;44(5):1127-49. https://doi.org/10.1016/j.foodres.2011.03.053

46. Huang S, Vignolles ML, Chen XD, Le Loir Y, Jan G, Schuck P, et al. Spray drying of probiotics and other food-grade bacteria: A review. Trends Food Sci Technol. 2017 May;63:1-17. https://doi.org/10.1016/j.tifs.2017.02.007

47. Peighambardoust SH, Golshan Tafti A, Hesari J. Application of spray drying for preservation of lactic acid starter cultures: a review. Trends Food Sci Technol. 2011 May;22(5):215-24. https://doi.org/10.1016/j.tifs.2011.01.009

48. Anal AK, Singh H. Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends Food Sci Technol. 2007 May;18(5):240-51. https://doi.org/10.1016/j.tifs.2007.01.004

49. Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV. Microencapsulation of probiotics for gastrointestinal delivery. J Controlled Release. 2012 Aug;162(1):56-67. https://doi.org/10.1016/j.jconrel.2012.06.003 PMid:22698940

50. De Vos P, Faas MM, Spasojevic M, Sikkema J. Encapsulation for preservation of functionality and targeted delivery of bioactive food components. Int Dairy J. 2010 Apr;20(4):292-302. https://doi.org/10.1016/j.idairyj.2009.11.008

51. Meng XC, Stanton C, Fitzgerald GF, Daly C, Ross RP. Anhydrobiotics: The challenges of drying probiotic cultures. Food Chem. 2008 Feb;106(4):1406-16. https://doi.org/10.1016/j.foodchem.2007.04.076

52. Perdana J, Fox MB, Schutyser MAI, Boom RM. Enzyme inactivation kinetics: Coupled effects of temperature and moisture content. Food Chem. 2012 Jul;133(1):116-23. https://doi.org/10.1016/j.foodchem.2011.12.080

53. Kailasapathy K. Microencapsulation of probiotic bacteria: technology and potential applications. Curr Issues Intest Microbiol. 2002 Sep;3(2):39-48. PubMed PMID: 12400637.

54. Martín MJ, Lara-Villoslada F, Ruiz MA, Morales ME. Microencapsulation of bacteria: A review of different technologies and their impact on the probiotic effects. Innov Food Sci Emerg Technol. 2015 Feb;27:15-25. https://doi.org/10.1016/j.ifset.2014.09.010

55. Yao M, Xie J, Du H, McClements DJ, Xiao H, Li L. Progress in microencapsulation of probiotics: A review. Compr Rev Food Sci Food Saf. 2020 Mar;19(2):857-74. https://doi.org/10.1111/1541-4337.12532 PMid:33325164

56. Krasaekoopt W, Bhandari B, Deeth H. Evaluation of encapsulation techniques of probiotics for yoghurt. Int Dairy J. 2003 Jan;13(1):3-13. https://doi.org/10.1016/S0958-6946(02)00155-3

57. Champagne CP, Fustier P. Microencapsulation for the improved delivery of bioactive compounds into foods. Curr Opin Biotechnol. 2007 Apr;18(2):184-90. https://doi.org/10.1016/j.copbio.2007.03.001 PMid:17368017

58. Dianawati D, Mishra V, Shah NP. Survival of Microencapsulated Probiotic Bacteria after Processing and during Storage: A Review. Crit Rev Food Sci Nutr. 2016 Jul 26;56(10):1685-716. https://doi.org/10.1080/10408398.2013.798779 PMid:25853290

59. Frakolaki G, Giannou V, Kekos D, Tzia C. A review of the microencapsulation techniques for the incorporation of probiotic bacteria in functional foods. Crit Rev Food Sci Nutr. 2021 May 15;61(9):1515-36. https://doi.org/10.1080/10408398.2020.1761773 PMid:32400195

60. Crowe JH, Carpenter JF, Crowe LM. THE ROLE OF VITRIFICATION IN ANHYDROBIOSIS. Annu Rev Physiol. 1998 Oct;60(1):73-103. https://doi.org/10.1146/annurev.physiol.60.1.73 PMid:9558455

61. Conrad PB, Miller DP, Cielenski PR, De Pablo JJ. Stabilization and Preservation of Lactobacillus acidophilus in Saccharide Matrices. Cryobiology. 2000 Aug;41(1):17-24. https://doi.org/10.1006/cryo.2000.2260 PMid:11017757

62. Leslie SB, Israeli E, Lighthart B, Crowe JH, Crowe LM. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol. 1995 Oct;61(10):3592-7. https://doi.org/10.1128/aem.61.10.3592-3597.1995 PMid:7486995 PMCid:PMC167656

63. Kurtmann L, Carlsen CU, Risbo J, Skibsted LH. Storage stability of freeze-dried Lactobacillus acidophilus (La-5) in relation to water activity and presence of oxygen and ascorbate. Cryobiology. 2009 Apr;58(2):175-80. https://doi.org/10.1016/j.cryobiol.2008.12.001 PMid:19111715

64. Capela P, Hay TKC, Shah NP. Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt. Food Res Int. 2006 Mar;39(2):203-11. https://doi.org/10.1016/j.foodres.2005.07.007

65. Iravani S, Korbekandi H, Mirmohammadi SV. Technology and potential applications of probiotic encapsulation in fermented milk products. J Food Sci Technol. 2015 Aug;52(8):4679-96. https://doi.org/10.1007/s13197-014-1516-2 PMid:26243890 PMCid:PMC4519473

66. Rokka S, Rantamäki P. Protecting probiotic bacteria by microencapsulation: challenges for industrial applications. Eur Food Res Technol. 2010 May;231(1):1-12. https://doi.org/10.1007/s00217-010-1246-2

67. Marcial-Coba MS, Knøchel S, Nielsen DS. Low-moisture food matrices as probiotic carriers. FEMS Microbiol Lett. 2019 Jan 1;366(2). https://doi.org/10.1093/femsle/fnz006

68. Champagne CP, Ross RP, Saarela M, Hansen KF, Charalampopoulos D. Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices. Int J Food Microbiol. 2011 Oct;149(3):185-93. https://doi.org/10.1016/j.ijfoodmicro.2011.07.005 PMid:21803436

69. Doron S, Snydman DR. Risk and Safety of Probiotics. Clin Infect Dis. 2015 May 15;60(suppl_2):S129-34. https://doi.org/10.1093/cid/civ085 PMid:25922398 PMCid:PMC4490230

70. Fredua-Agyeman M, Gaisford S. Comparative survival of commercial probiotic formulations: tests in biorelevant gastric fluids and real-time measurements using microcalorimetry. Benef Microbes. 2015 Jan 1;6(1):141-52. https://doi.org/10.3920/BM2014.0051 PMid:25351484

71. Morovic W, Hibberd AA, Zabel B, Barrangou R, Stahl B. Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases. Front Microbiol. 2016;Volume 7-2016. https://doi.org/10.3389/fmicb.2016.01747 PMid:27857709 PMCid:PMC5093124

72. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. (2003). ICH harmonised tripartite guideline Q1A(R2): Stability testing of new drug substances and products. https://www.ich.org 

73. Santivarangkna C, Kulozik U, Foerst P. Alternative Drying Processes for the Industrial Preservation of Lactic Acid Starter Cultures. Biotechnol Prog. 2007 Jan;23(2):302-15. https://doi.org/10.1021/bp060268f PMid:17305363

74. Misra S, Pandey P, Mishra HN. Novel approaches for co-encapsulation of probiotic bacteria with bioactive compounds, their health benefits and functional food product development: A review. Trends Food Sci Technol. 2021 Mar;109:340-51. https://doi.org/10.1016/j.tifs.2021.01.039

75. Almeida A, Nayfach S, Boland M, Strozzi F, Beracochea M, Shi ZJ, et al. A unified catalog of 204,938 reference genomes from the human gut microbiome. Nat Biotechnol. 2021 Jan;39(1):105-14. https://doi.org/10.1038/s41587-020-0603-3 PMid:32690973 PMCid:PMC7801254

76. Wendel U. Assessing Viability and Stress Tolerance of Probiotics-A Review. Front Microbiol. 2022 Jan 27;12:818468. https://doi.org/10.3389/fmicb.2021.818468 PMid:35154042 PMCid:PMC8829321

77. Wilkinson MG. Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review. Trends Food Sci Technol. 2018 Aug;78:1-10. https://doi.org/10.1016/j.tifs.2018.05.006