Cell immobilization techniques for the preservation of probiotics

Abstract

Incorporation of probiotic cultures in products in order to replenish or supplement the normal gastrointestinal microflora is a well known and accepted practice. However survival of these cultures is a problem due to a number of reasons including effects of storage conditions. Various researchers from different countries around the world have reported probiotic product instability. Microencapsulation has been used in an attempt to solve this problem. However, most methods involve the use of organic solvents which is not ideal because their toxicity may cause destruction of the microbial cells. A novel encapsulation method for probiotics, which excludes the use of organic solvents, was developed by the Council for Scientific and Industrial Research (CSIR) (US Patent Application no. 20050112205). This thesis investigated the efficiency/potential of this new method for increasing stability of sensitive probiotic cultures, specifically bifidobacteria. Early studies using both culture dependent and culture independent techniques showed reduced numbers of viable cultures in probiotic products, mainly yoghurts, from all around the world. These results were confirmed in this study for similar products sold in South Africa. Most of the product labels did not specify viable numbers of probiotics nor the identity (genus and species names) of the microorganisms incorporated. Successful encapsulation of bifidobacteria was achieved using the CSIR patented method. Complete encapsulation was indicated by absence of cells on surfaces of the encapsulated particles and production of a product with an acceptable particle size distribution was obtained. It was also demonstrated that the encapsulation process produced no visible morphological changes to the bacterial cells nor did it have a negative effect on cell viability over time. The potential of interpolymer complex formation in scCO2 for the encapsulation of sensitive probiotic cultures was demonstrated for the first time. Once ingested, probiotic cultures are exposed to unfavourable acidic conditions in the upper gastrointestinal tract. It is desired that these cultures be protected from this in order to increase the viability of the probiotics for efficient colonization. Interpolymer complex encapsulated B. longum Bb-46 cells were therefore exposed to simulated gastric fluid (SGF) and subsequently to simulated intestinal fluid (SIF). It was found that the interpolymer complex protected bifidobacteria from gastric acidity, displaying pH-responsive release properties, with little to no release in SGF and substantial release in SIF. Thus the interpolymer complex demonstrated desirable characteristics retaining the encapsulated bacteria inside when conditions were unfavourable and only releasing them under favourable conditions. Survival was improved by the incorporation of glyceryl monostearate (GMS) in the matrix and by use of gelatine capsules. Protection efficiency of the interpolymer matrix was better when higher loading of GMS was used. Use of polycaprolactone (PCL) as an alternative to poly (vinylpyrrolidone) (PVP) and incorporation of ethylene oxide-propylene oxide triblock copolymer (PEO-PPO-PEO) affected the interpolymer complex negatively, rendering it swellable in the low pH environment exposing the bifidobacteria to gastric acidity. The use of beeswax seemed to have a more protective effect though results were inconclusive. Probiotic cultures must also remain viable in products during storage. Encapsulated bacteria were either harvested from the reactor after 2 h of equilibration followed by depressurization, and then ground to a fine powder or after 2 h of equilibration the liquefied product was sprayed through a capillary tube with a heated nozzle at the end, into the product chamber. Encapsulated bacteria were stored in either sterile plastic bags or glass bottles under different conditions and then viable counts were determined over time. Survival of bacteria was generally better when the products were stored in glass bottles than in plastic bags. Bacteria encapsulated in an interpolymer complex formed between PVP and vinyl acetate-crotonic acid copolymer (VA-CA), (PVP:VA-CA) survived better than non-encapsulated bacteria under all storage conditions when the product was recovered from the reaction chamber. When the product was recovered from the product chamber, numbers of viable non-encapsulated bacteria were higher than the encapsulated bacteria for all interpolymer complex formulations. This was probably due to some exposure to high shear during spraying into the product chamber. The interpolymer complex between PCL and VA-CA i.e. PCL:VA-CA seemed weaker than the PVP:VA-CA nterpolymer complex as viable counts of bacteria released from it were lower than those from the latter complex. Addition of PEO-PPO-PEO to both the PVP:VA-CA and PCL:VA-CA complexes decreased the protection efficiency. However, results indicated that sufficient release of encapsulated bacteria from the interpolymer complexes was obtained when the encapsulated material was incubated in SIF rather than in Ringer’s solution. When SIF was used for release of encapsulated bacteria, the shelf life of B. longum Bb-46 was doubled. Encapsulation in an interpolymer complex therefore provided protection for encapsulated cells and thus has potential for improving shelf life of probiotic cultures in products. Further studies will investigate the effects of encapsulating probiotics together with prebiotics in the interpolymer complex as well as effects of encapsulating combinations of different probiotic strains together, both on survival in simulated gastrointestinal tract and during storage. The unique particles produced using the patented encapsulation technique increased the stability of probiotic cultures. This technique may find significant application in industries manufacturing probiotic products, especially food and pharmaceuticals, thereby improving the well being of consumers.