MITU Mirela Adriana1, OZON Emma Adriana1*, LUPULIASA Dumitru1, BALACI Teodora Dalila1, NIȚULESCU Georgiana1, POPESCU Ioana1, NICOARĂ Anca1, KARAMPELAS Oana1
1 Pharmaceutical Technology Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)
*corresponding author, email@example.com
Due to their large potential applications in human diseases such as gastrointestinal disorders, immunomodulation or cholesterol reduction, probiotic products are of great interest to the pharmaceutical industry. The formulation of the pharmaceutical forms containing probiotics aims the ability of the final product to protect the active ingredients from the harsh conditions and the delivery of the bacteria into the intestinal tract to assure their therapeutically effects.
Key words: probiotics, stability, pharmaceutical products
According to World Health Organization the probiotics are viable live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host . The amounts of probiotics in a product is expressed in Colony Forming Units (CFUs), and the declared quantity reflects the quantity of live microorganisms at the end of the stated shelf life, not at the time of manufacture.
In order to provide the target dose until end of shelf life and to compensate for potential losses during storage and handling, an overage is commonly included in the product [2,3]. The stability of probiotics, not only in terms of viability, but also in terms of metabolic and functional activity, is needed to provide the claimed health benefit during the whole shelf-life of the product. The application of different methods to increase probiotic stability and functionality has been the subject of many studies [4,5,6,7].
More stability testing guidelines were established to ensure that the stated shelf life of a given probiotic product is scientifically supported. Storage recommendations were provided to facilitate the communication of storage and handling instructions to customers.
Probiotics used as active pharmaceutical ingredients
The most commonly strains are Lactobacillus species like L. acidophilus, L. johnsonii, L. casei, L. rhamnosus, L. gasseri and L. reuteri. Bifidobacterium strains include B. bifidum, B. longum, B. lactis, B. infantis. However, Saccharomyces boulardii, Escherichia coli and enterococcus strains can also be used as probiotics . In order to obtain a high-performance pharmaceutical product, it must be well established the bacteria’s nutritional requirements and developing a tailored fermentation medium that supports growth and enhances the ability of the cells to survive and adjust to the stresses imposed by the manufacturing process .
The viability of microbial biomass is assured by using some dehydration methods like: freeze-drying, sublimation drying, including fluidization drying using different carriers and spray drying . The preferred drying method for thermally sensitive bacteria is freeze drying as it leads to the highest level of survival. The disadvantages of this method are: the long drying time and the fact that is expensive, but for drying of starter cultures, spray drying can be a viable alternative if the survival can be raised to make it economically attractive .
Also, encapsulation of probiotics is used to increase the bacteria resistance to freezing and freezing drying. In most of the studies, the probiotics were included in a gel matrix of biological nature materials such as alginate, k-carrageenan and gellan/xanthan [12,13]. Microencapsulation may improve the survival of these microorganisms, during both processing and storage, and also during passage through the human gastrointestinal tract.
Pharmaceutical products containing probiotics
The probiotics are included in different pharmaceutical forms, such as: powders, capsules, tablets, lozenges, suspensions, pessaries, etc. Most of them are oral delivery systems, as the targeted site is the colon. Capsules are representing the most used dosage form for probiotics because they allow for 2 major differentiators combined into one form: higher potency and longer stability.
Products performance depends on the physical and chemical properties of the excipients [14,15]. Most commonly used excipients are microcrystalline cellulose or rice maltodextrin (as binders and fillers), magnesium stearate and silicon dioxide (as lubricants), cellulose derivates (as suspending, viscosity agent), etc [16,17,18,19,20,21]. Lately, mannitol become widely used. It has the property of excellent stability, good flowability, low hygroscopicity, good solubility in different solvents, low caloric values and that is why it is reported as a better excipient for active ingredients which are moisture sensitive [22,23]. It was reported that the excipients like lactose, ascorbic acid and inulin have a positive impact on the stability of formulation using probiotic organism Pediococcus sp. .
Many different conditions present during the manufacture and storage of the product may affect the stability of probiotics: temperature, pH, water activity (aw), oxygen content or the presence of chemicals, and other microorganisms . The manufacturing of the pharmaceutical product must be happen under strict temperature and humidity controlled conditions. High compression force during manufacturing reduces the viability of probiotics in the tablets . The formulation of the pharmaceutical form containing probiotics aims the ability of the final product to protect the active ingredients from the harsh conditions and the delivery of the bacteria into the intestinal tract to assure their therapeutically effects.
Klayraunga et. al. designed a tablets formulation for probiotics that is protecting them from the degradation at low pH and deliver them to the intestinal tract in a viable form. Hydroxypropyl methylcellulose phthalate was used as a matrix due to the fact that it is insoluble in gastric fluids (pH ~1.5, but it rapidly dissolves in the upper small intestine where the pH is around ~ 5.5 .
The prospective area of probiotic stability and delivery improvement includes use of other excipients which are suitable for the process as well as provide biocompatibility and cause less cell viability. Incorporation of other process technologies to combat the process related issue, such as use of cryoprotectants in the mixture .
Packaging of pharmaceutical products containing probiotics
Pharmaceutical products containing probiotics must be packaged and handled in order to avoid the unfavorable influence of the medium factors, such as oxygen, warmth or moisture. Usually, it is considered that the refrigeration will prolong the potency and viability of most probiotics to maintain higher amounts over a longer period of time. Protective factors that help to preserve the freshness and viability of the probiotic strains in a supplement include refrigeration, resistant packaging, and storage in a cool, dry place [28,29]. The package for storage plays an important role because oxygen may affect a number of active probiotic cells and thus trigger the reduction of cell survivability over the entire storage period [30,31].
Glass, a chemically inert, non-porous, rigid packaging material can give almost 100% barrier against moisture and oxygen transmission. Among the flexible packaging, aluminium foil of different thickness gives protection against moisture and oxygen as compared to glass. Moisture barrier properties of extrusion-coated polymers also play an important role. The general selection of moisture barrier polymers includes low and high density polyethylene (LDPE-HDPE), polypropylene (PP), and polyethylene terephthalate. These also include cyclo-olefine copolymers, metallocene, nanocomposites etc .
Studies performed in our research laboratory
Over time, we developed many studies which aimed the formulation of different solid pharmaceutical products containing probiotics as active ingredients. We obtained capsules, conventional tablets, tablets for oral dispersion, powders and chewable tablets. As probiotic strains we used either Lactobacillus species, either Bifidobacterium strains, or a mixture of these species.
Our first challenge was always the weak flowability of the composed powders due to the used active ingredients. Although the selected excipients present excellent flowing and compressibility properties, when the probiotics are added we can notice a drastic diminishing of these characteristics, the final powders presenting a weak flowability, a low to medium compressibility, a smaller particle size and higher moisture content due to the probiotics hygroscopicity. Regardless the selected probiotic strains, the compound powder needed the adding of three lubricants: talcum, magnesium stearate and silicon dioxide.
The flowability, determined with an Automated Powder and Granulate Testing System PTG-S3 (Pharma Test Apparatebau GmbH, Germany), varied as follows: the flowing time was between 30 seconds (for powders containing Lactobacillus species) and 60 seconds (for powders containing mixture of species), the angle of repose varied from 35° to 52° (for the same powders), and the flowing rate was around 1 g/s, values which prove that the powders have low free flow which can be increased if stirring is used during the tests.
Regarding the volumetric characteristics – Hausner and Carr index, performed with Vankel Tap Density Tester (Vankel Industries Inc., USA), Hausner ratio varied between 1,56 and 1,78, and Carr index from 26% to 41%, showing us that the compound powders present a medium to low compressibility, fact which makes the powder difficult to be processed under the form of tablets.
The distribution of particle size on granulometric classes tested by the sieving method, using a CISA Sieve Shaker Mod. RP 10 (Cisa Cedaceria Industrial, Spain) indicated us that a significant part of particles have small sizes (less than 160 μm). Also, the loss on drying can provide us meaningful information about the behavior of the powder during processing under solid pharmaceutical forms. We determined it by the Karl Fisher method with a Mettler Toledo DL 35 apparatus, and the registered results were between 3 and 5% moisture in the powders.
It could be concluded that the pharmaco-technical properties of the powders are much influenced by the probiotics included even in low doses, and also by the type of strains, the best results being obtained when Lactobacillus species alone, where used.
The excipients where selected considering the type of pharmaceutical form we wanted to obtain, the flowing and compressing characteristics established in the pre-formulation phase and by the desirable manufacturing method. For the manufacturing of powders or capsules the excipients influence is only on the flowing properties, and the problems were solved by using fillers with very good flowability (like microcrystalline cellulose) and combining the three mentioned lubricants in the powder mixtures. For tablets manufacturing we have always used the direct compression method, a very fast technology, in order to ensure the probiotics stability by not using in any stages high humidity or high temperatures. We selected different direct compressible excipients and the best results were found when using Flowlac (spray-dried lactose) for conventional tablets, and F-Melt (provided by Fuji Chemical Industries Co., Ltd., Japon) which contains carbohydrates, disintegrants and inorganic ingredients or F-Melt combined with CompactCel (produced by BIOGRUND GmbH, Germany) which is a mixture of polyols and cellulose fibers with a dry binder and taste masking roles.
One of the most significant aspects which must be considered during manufacturing process, which acts upon the quality and the stability of the pharmaceutical products and must be well established is the necessary compression force. In our studies we used three type of compression forces: low (around 10 kN), medium (approximately 15 kN) and high (above 20 kN). When using high forces, the obtained tablets showed an unsatisfying high friability (more than 2%) with low mechanical resistance (between 20 and 40 N), and also the stability decreased critically. Meantime, after a low or medium compression force, the friability and hardness registered values within the limits imposed by rules into force, with fast disintegration time, and a good stability.
All the manufactured products were exposed to stability tests according to the guidelines under different storage conditions: long term (12 months) at 25ºC±2ºC/60%RH±5%RH, intermediate (6 months) at 30ºC±2ºC/65%RH±5%RH and accelerated (6 months) at 40ºC±2ºC/75%RH±5%RH. The characteristics of the products that were followed are: pharmaco-technical properties (flow rate, moisture content, average weight, friability and hardness), biopharmaceutical characteristics (disintegration time, release rate) and viability of the contained probiotics.
In addition to having beneficial health effects, probiotic bacteria must possess the properties required to be used as pharmaceutical products, such as safety, stability, ability to be produced on a large scale and viability [32-35]. One of the most important criteria is the maintenance of viability during the product life cycle  which can be affected by the culture medium, manufacturing process and residual humidity . It is essential to well-known probiotic strain’s technological properties. They are usually selected based on this aspect. Also, when a mixture of probiotics is chosen, should be considered the antagonistic relationship between them.
As mentioned before, there are a lot of factors during production, processing and storage that affect the amount of viable or active probiotics cells that can induce the therapeutically effects . They include products parameters (pH, molecular oxygen, water activity, presence of other ingredients, flavors and coloring agents); processing parameters (heat treatment, incubation temperature, cooling rate, packaging materials and storage methods); microbiological parameters (strains of probiotics, rate and proportion of inoculation). In addition, the presence of oxygen can cause formation and accumulation of toxic metabolites in cells [39-41], which can lead to cell death by oxidative damage [6,42].
There are a lot of challenges for pharmaceutical technology when manufacturing solid dosage forms containing probiotics starting with the preformulation studies, completing with manufacturing methods and concluding with the stability and efficacy studies.
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