Probiotics in dietary supplements – how the choice of capsule shell and lyophilizate form affects CFU survival

The label of a probiotic supplement may declare, for example, 10^9 CFU per capsule. The question that is rarely asked directly is: is this number accurate when the consumer opens the package after several or a dozen months, or does it describe the status only at the time of production? This distinction is of fundamental importance because a probiotic is a live product, and live bacterial cells lose viability over time.

CFU (colony forming units) is the only objective measure of probiotic quality. Without a sufficient number of live microorganisms, the product does not meet the definition of a probiotic adopted by international scientific consensus (Hill et al., 2014). The CFU count is not a fixed characteristic – it degrades under the influence of moisture, oxygen, temperature, and gastric acid.

For a company outsourcing contract manufacturing, the choice of capsule form, shell material, and storage conditions is not a finishing detail, but factors that directly affect the CFU stability test result and the credibility of the declaration placed on the label. Below, we explain exactly what determines how many live bacteria reach the consumer’s body.

What is a probiotic and why “live” is the only quality criterion

The International Scientific Association for Probiotics and Prebiotics (ISAPP), in its 2014 consensus statement, defined a probiotic as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”, i.e. live microorganisms which, when administered in appropriate amounts, provide a health benefit to the host (Hill et al., 2014). Two elements of this definition are key from a production perspective: “live” and “adequate amounts”.

CFU, or colony forming unit, is a single live microorganism capable of dividing and forming a visible colony on a culture medium. It is an operational measure of viability: only those cells that can multiply are counted, not dead or damaged cells. The result is expressed as CFU per gram or CFU per capsule. Importantly, the value measured during production is not equal to the value at the end of the shelf life, because part of the bacterial population dies during that time.

This leads to a practical consequence. The “adequate amounts” element in the ISAPP definition means that the number of live microorganisms must be sufficient at the time of consumption, not merely at the time of manufacture. If a supplement declares 10^9 CFU per capsule, this value should be confirmed through to the end of the shelf life, when stored in accordance with the recommendations on the label. To achieve this, the manufacturer typically assumes an overage of live cells from the outset, usually 10- to 100-fold relative to the declared dose, in order to cover the inevitable CFU losses over time (Rajam and Subramanian, 2022).

Four main factors degrading the viability of probiotic strains

The survival of lyophilized probiotic strains depends on several well-described environmental factors. Their control at the formulation, packaging, and storage stages determines how many live cells will survive until the end of the shelf life (Agriopoulou et al., 2023; Rajam and Subramanian, 2022).

• Moisture (water activity, aw) – the most dangerous factor for lyophilized strains. Water activity above 0.3 rapidly accelerates the loss of cell viability. The production target is to maintain aw below 0.3 in the finished, closed, and packaged capsule (Agriopoulou et al., 2023);
• Oxygen – sensitive strains, particularly those of the genus Bifidobacterium, are anaerobes or microaerophiles. Even a small amount of oxygen can damage their lipid membranes. Capsules with low oxygen permeability and nitrogen flushing provide additional protection;
• Gastric acid (pH 1.2-2.0) – most strains die within several dozen minutes at a pH below 3. Acid-sensitive strains, such as Lactobacillus acidophilus or Bifidobacterium longum, require active protection during transit through the stomach;
• Temperature – storage at room temperature (25°C) causes a significantly faster loss of CFU than storage under refrigerated conditions (4°C). Products declared as stable at room temperature must be based on strains with documented thermotolerance or on appropriate protective matrices (Rajam and Subramanian, 2022).

These four factors act together and reinforce one another. Higher temperature accelerates reactions triggered by moisture, while the presence of oxygen deepens oxidative damage to cell membranes. From the manufacturer’s perspective, this means that protecting CFU survival does not come down to a single solution, but to consistent management of the entire chain: strain selection, protective matrix, capsule shell, and outer packaging.

Worth knowing: water activity (aw) and why the 0.3 threshold is critical for probiotics

Water activity (aw) is a measure of the availability of water for chemical and biological reactions – it is not the same as percentage moisture content. An aw value of 1.0 corresponds to pure water, while aw = 0 means a complete absence of available water. Lyophilized probiotic strains show a dramatic decrease in viability once the aw = 0.3 threshold is exceeded: water triggers enzymatic and chemical reactions leading to damage to cell membranes and bacterial DNA (Agriopoulou et al., 2023). For this reason, a capsule intended for probiotics should not introduce additional moisture into the fill. It is precisely this criterion that differentiates shell materials and makes them an important element in designing a probiotic product.

How capsule shell material affects CFU during storage

The capsule shell itself does not directly affect bacterial viability. Its role is indirect, but real: it determines how much moisture the shell material introduces into the inside of the capsule or absorbs during storage. Because a lyophilized probiotic is hygroscopic and the critical aw = 0.3 threshold is low, every additional source of water in the immediate vicinity of the strain matters. Differences in the natural moisture content between hard capsule shell materials are well documented in this respect (Yang et al., 2020).

Gelatin (13-16% moisture)

A gelatin shell is characterized by a high natural water content, in the range of 13-16% (Yang et al., 2020). This represents a potential source of additional moisture for the hygroscopic lyophilizate contained inside it. During long-term storage, especially under conditions of elevated temperature or relative humidity, water from the shell may migrate into the capsule fill. For strains sensitive to exceeding the aw = 0.3 threshold, this effect may accelerate the loss of viability and thereby reduce the CFU stability test result.

HPMC (4.5-6.5% moisture)

Hydroxypropyl methylcellulose (HPMC) capsules have a significantly lower moisture content, in the range of 4.5-6.5% (Yang et al., 2020). The lower water content in the shell minimizes the risk of moisture migration into the lyophilized fill. The cited study indicated that “HPMC capsules have weaker moisture sorption ability and moisture keeping ability than pullulan or gelatin capsules”, meaning that HPMC capsules have a weaker capacity to absorb and retain moisture than pullulan or gelatin capsules (Yang et al., 2020). In addition, HPMC is not subject to cross-linking, which eliminates the problem of prolonged disintegration time during storage. For these reasons, HPMC is the preferred choice for probiotics among standard hard capsules.

DRcaps (delayed-release HPMC capsules)

DRcaps-type capsules are a form of HPMC capsules designed to slow the release of contents at low pH. Studies have shown their superior protection of acid-sensitive strains during gastric transit compared with standard HPMC capsules (Agriopoulou et al., 2023). However, terminological precision must be maintained: delayed release in DRcaps capsules is not the same as a pharmacopeial enteric coating. It may offer practical protection for sensitive strains, but where regulatory requirements concerning full resistance to gastric acid apply, a proper enteric coating with verification by a dissolution test is necessary.

Worth knowing: CFU “at production” versus CFU “at the end of shelf life” – what to declare

The industry standard is to declare CFU at the end of shelf life, not the value from the time of production. Only this approach guarantees that the consumer receives the declared dose of live bacteria throughout the entire product shelf life. In practice, this means that the manufacturer must produce the product with a CFU overage, typically 10- to 100-fold relative to the declared value, in order to cover the inevitable loss of viability over time (Rajam and Subramanian, 2022). Three variables determine how large the production overage needs to be and what expiry date can be credibly declared: strain selection together with its natural thermotolerance and resistance to storage conditions, the protective matrix composed of cryoprotectants, and the capsule shell material.

Protective matrix and cryoprotectants – what is not visible on the supplement label

The capsule is only the final element of live cell protection. Before that, the lyophilized strain should be protected by an appropriately selected protective matrix, i.e. a set of cryoprotectants that prevent cell damage during the lyophilization process itself and during subsequent storage (Rajam and Subramanian, 2022). This is why lyophilization (freeze-drying) is considered the most effective method for preserving probiotic viability: it makes it possible to obtain a dry product with water activity below 0.1 immediately after the process, provided that the proper protective materials are used.

In addition to lyophilization, probiotic technology also uses other encapsulation and preservation techniques, such as spray-drying, emulsion, or granulation, but lyophilization remains the method with the best CFU preservation profile (Rajam and Subramanian, 2022). The most commonly used cryoprotectants include:

• trehalose – a disaccharide considered one of the most effective cryoprotectants, stabilizing cell membranes during drying and storage;
• skimmed milk – a classic protective matrix; it contains lactose and proteins that perform a protective function for bacterial cells;
• inulin (FOS) – a prebiotic that protects cells during lyophilization and may additionally serve as a nutritional substrate for bacteria; s
• modified starch – a filler with protective properties, facilitating the achievement of an appropriate lyophilizate structure.

Internal shell materials used in micro- and macroencapsulation techniques that protect bacteria against moisture and gastric acid include, among others, alginate, HPMC, chitosan, and gelatin (Agriopoulou et al., 2023). The conclusion for the manufacturer is clear: the CFU declaration on the label has real value only when it is supported by a properly designed lyophilization process, a selected protective matrix, and the right choice of capsule shell. Omitting any of these layers increases the rate of viability loss and undermines the credibility of the declared dose.

Plan your probiotic formulation with Eubioco

Strain selection, determination of the starting CFU concentration, design of the protective matrix, and choice of capsule type are decisions that should be made together, not sequentially. Each of them affects the others and the final stability test result. Eubioco, as a contract manufacturer operating in accordance with GMP and ISO 22000:2018 standards, manufactures probiotic supplements in dry solid forms. Contact us to discuss your project.

Bibliography

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2. Agriopoulou, S. et al. (2023). Application of Encapsulation Strategies for Probiotics: From Individual Loading to Combinations of Encapsulants for Enhanced Protection. Microorganisms, 11, 2896. DOI: 10.3390/microorganisms11122896. [online] Available at: https://doi.org/10.3390/microorganisms11122896 [accessed online: 20.05.2026]

3. Rajam, R., Subramanian, P. (2022). Encapsulation of probiotics: past, present and future. Beni-Suef University Journal of Basic and Applied Sciences, 11(1). DOI: 10.1186/s43088-022-00228-w. [online] Available at: https://doi.org/10.1186/s43088-022-00228-w [accessed online: 20.05.2026]

4. Yang, N., Chen, H., Jin, Z., Hou, J., Zhang, Y., Han, H., Shen, Y., Guo, S. (2020). Moisture sorption and desorption properties of gelatin, HPMC and pullulan hard capsules. International Journal of Biological Macromolecules, 159, pp. 659-666. DOI: 10.1016/j.ijbiomac.2020.05.110. [online] Available at: https://doi.org/10.1016/j.ijbiomac.2020.05.110 [accessed online: 20.05.2026]