Survival of Microencapsulated Probiotic Bacteria after Processing and during Storage: A Review

The use of live probiotic bacteria as food supplement has become popular. Capability of probiotic bacteria to be kept at room temperature becomes necessary for customer's convenience and manufacturer's cost reduction. Hence, production of dried form of probiotic bacteria is important. Two common drying methods commonly used for microencapsulation are freeze drying and spray drying. In spite of their benefits, both methods have adverse effects on cell membrane integrity and protein structures resulting in decrease in bacterial viability. Microencapsulation of probiotic bacteria has been a promising technology to ensure bacterial stability during the drying process and to preserve their viability during storage without significantly losing their functional properties such acid tolerance, bile tolerance, surface hydrophobicity, and enzyme activities. Storage at room temperatures instead of freezing or low temperature storage is preferable for minimizing costs of handling, transportation, and storage. Concepts of water activity and glass transition become important in terms of determination of bacterial survival during the storage. The effectiveness of microencapsulation is also affected by microcapsule materials. Carbohydrate- and protein-based microencapsulants and their combination are discussed in terms of their protecting effect on probiotic bacteria during dehydration, during exposure to harsh gastrointestinal transit and small intestine transit and during storage.     

Spray‐drying for the production of dried cultures

This review emphasises the importance of spray‐drying as an under‐used but promising technique to preserve viable and active starter cultures and also, potentially, probiotic cultures. The knowledge concerning the production of spray‐dried starter cultures is discussed. Different drying techniques and micro‐organisms have been investigated for their survival through the drying process. During drying and subsequent storage in the dried state, bacteria are subjected to several stresses, which have already been described as causing damage to cells, leading to the loss of cellular viability and activity. Some studies found that several factors/strategies can confer improved cellular viability.     

A feasibility study of Lactobacillus plantarum in fruit powders after processing and storage

The aim of this study was to develop fruit powders (apple, banana and strawberry) enriched with a probiotic strain (Lactobacillus plantarum 299v). Two methodologies were proposed: (i) drying of the fruit with probiotic culture incorporated (by convection) or (ii) drying of fruit (by convection) and addition of spray‐dried probiotic culture. In the first methodology, processing caused a notable reduction in probiotic viable counts in apple, but this reduction was lower during drying of banana and strawberry. A large reduction in viable cells was also recorded during storage. In the second methodology, the survival of L. plantarum 299v was considerably higher during spray‐drying, and fruit powders with a microbial content suitable for a probiotic food (10⁸–10⁹ cfu g^?1) were obtained. The fruit powders incorporating L. plantarum 299v can be stored at 4 °C or at room temperature, for at least 3 months. This preliminary study demonstrated that fruit powders are good carriers of probiotic cultures, but the techniques used to produce them should be carefully considered.     

Comparative survival and evaluation of functional probiotic properties of spray-dried lactic acid bacteria

The effect of spray drying on the viability and retention of key probiotic properties like acid and bile tolerance and cholesterol assimilation of three probiotic lactic acid bacteria (LAB) ( Lactobacillus plantarum CFR 2191, Lactobacillus salivarius CFR 2158 and Pediococcus acidilactici CFR 2193) has been studied. More than 97% survival was exhibited by the three LAB (1% cell suspension) spray dried with maltodextrin and nonfat skimmed (NFSM) as carriers. LAB cultures spray dried with maltodextrin showed significantly greater ( P ≤  0.05) retention of functional properties than those with NFSM. The results highlight a cost-effective way of producing large quantities of selected probiotic cultures with functional properties for neutraceutical application.     

Protective approaches and mechanisms of microencapsulation to the survival of probiotic bacteria during processing,storage and gastrointestinal digestion: A review

Abstract

In recent years, there is a rising interest in the number of food products containing probiotic bacteria with favorable health benefit effects. However, the viability of probiotic bacteria is always questionable when they exposure to the harsh environment during processing, storage, and gastrointestinal digestion. To overcome these problems, microencapsulation of cells is currently receiving considerable attention and has obtained valuable effects. According to the drying temperature, the commonly used technologies can be divided into two patterns: high temperature drying (spray drying and fluid bed drying) and low temperature drying (ultrasonic vacuum spray drying, spray chilling, electrospinning, supercritical technique, freeze drying, extrusion, emulsion, enzyme gelation, and impinging aerosol technique). Furthermore, not only should the probiotic bacteria maintain high viability during processing but they also need to keep alive during storage and gastrointestinal digestion, where they additionally suffer from water, oxygen, heat as well as strong acid and bile conditions. This review focuses on demonstrating the effects of different microencapsulation techniques on the survival of bacteria during processing as well as protective approaches and mechanisms to the encapsulated probiotic bacteria during storage and gastrointestinal digestion that currently reported in the literature.     

Role of glassy state on stabilities of freeze-dried probiotics

High viability of dried probiotics is of great importance for immediate recovery of activity in fermented foods and for health-promoting effects of nutraceuticals. The conventional process for the production of dried probiotics is freeze-drying. However, loss of viability occurs during the drying and storage of the dried powder. It is believed that achieving the "glassy state" is necessary for survival, and the glassy state should be retained during freezing, drying, and storage of cells. Insight into the role of glassy state has been largely adopted from studies conducted with proteins and foods. However, studies on the role of glassy state particularly with probiotic cells are on the increase, and both common and explicit findings have been reported. Current understanding of the role of the glassy state on viability of probiotics is not only valuable for the production of fermented foods and nutraceuticals but also for the development of nonfermented functional foods that use the dried powder as an adjunct. Therefore, the aim of this review is to bring together recent findings on the role of glassy state on survival of probiotics during each step of production and storage. The prevailing state of knowledge and recent finding are discussed. The major gaps of knowledge have been identified and the perspective of ongoing and future research is addressed.     

Survival of free and encapsulated probiotic bacteria and their effect on the sensory properties of yoghurt

The survival and effect of free and calcium-induced alginate-starch encapsulated probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium lactis) on pH, exopolysaccharide production and influence on the sensory attributes of yogurt were studied over 7 weeks storage. Addition of probiotic bacteria (free or encapsulated) reduced acid development in yogurt during storage. Post-acidification in yogurt with encapsulated probiotic bacteria was slower compared to yogurt with free probiotic bacteria. More exopolysaccharides were observed in yogurts with probiotic cultures compared to those without probiotic cultures. The results showed that there was an increased survival of 2 and 1 log cell numbers of L. acidophilus and B. lactis, respectively due to protection of cells by microencapsulation. The addition of probiotic cultures either in the free or encapsulated states did not significantly affect appearance and colour, acidity, flavour and after taste of the yogurts over the storage period. There were, however, significant differences (P<0.05) in the texture (smoothness) of the yogurts. This study has shown that incorporation of free and encapsulated probiotic bacteria do not substantially alter the overall sensory characteristics of yogurts and microencapsulation helps to enhance the survival of probiotic bacteria in yogurts during storage.     

Foam‐Mat Freeze‐Drying of Bifidobacterium longum RO175: Viability and Refrigerated Storage Stability

Foaming as a pretreatment was used prior to freeze‐drying of Bifidobacterium longum RO175 to investigate the potential acceleration of the drying rate and increase in microorganism viability after the process. A study on storage of foamed and nonfoamed freeze‐dried products at 4 °C completed this study. B. longum RO175 in foamed medium could be freeze‐dried in 1/7 to 1/4 of the time required for nonfoamed suspensions. In addition, foamed suspensions presented higher viability immediately after freeze‐drying (13.6% compared to 12.81 % or 11.46%, depending on the cryoprotective media). Refrigerated storage led to a reduction in B. longum RO175 viability for all tested protective agents (foamed and nonfoamed). No correlation between glass transition temperature and stability of probiotic powders was observed during storage. In addition, lower viability after 56 d of storage was observed for foamed materials, probably due to foam porous structure and higher hygroscopicity, and oxygen presence and moisture pickup during storage.     

Proposal of a new reference method to determine the water content of dried dairy products

The water content of milk powders is “officially” determined by drying. The result of drying techniques is, however, the mass loss the sample undergoes under the conditions applied. No differentiation between water and other volatile substances is made. A particular problem is the lactose that is present in all dried dairy products. The α-form contains one mole of water of crystallisation per mole. This water fraction is not determined completely by ordinary drying methods. How much of the crystallised water is detected depends very much on the conditions applied. The entity determined by drying is not defined. It is only a tradition to regard the mass loss thus detected as water content. More and more, however, the expression moisture content is used for the result of drying techniques, although this term is also unsatisfactory and misleading. The discrepancy between mass loss on drying and water content becomes more pronounced when the product has high crystalline lactose content, such as whey powders or lactose itself. In these cases the difference between mass loss and water content can be quite significant. The existing reference method for moisture determination in dried milk is therefore not reasonably applicable on dairy products with high crystalline lactose content. It could be shown that the water content of dried dairy products can be determined using the Karl Fischer titration independently of the level of crystallisation water. The results are not only more accurate but also more precise than those obtained by the reference drying method. It is therefore proposed to introduce the Karl Fischer titration as reference method for dried dairy products.     

Microwave-Vacuum Drying of Lactic Acid Bacteria: Influence of Process Parameters on Survival and Acidification Activity

Freeze drying is one important step in downstream processing of probiotics and starter cultures production and requires a long duration of the drying step, which, therefore, often is the overall production bottleneck. Microwave accelerated vacuum drying already showed promising results in terms of product quality for various food products. However, poor information on drying microorganisms by microwaves is available. The aim of this work was to set up a suitable microwave-vacuum drying process for the conservation of lactic acid bacteria and to investigate the optimum process parameters to achieve dried cultures with high survival and activity. The probiotic Lactobacillus paracasei ssp. paracasei F19 was used as model strain. The influence of the process parameters chamber pressure (7–30 mbar), specific microwave power input (1–5 W/g), and maximum product temperature (30–45 °C) on the main quality parameters survival rate, metabolic activity, and water activity of the dried samples were analyzed. Continuous energy input was shown to act detrimentally to the cells due to extreme temperature rise in the second drying stage. Proper temperature regulation could be obtained by pulsed microwave input according to a maximum set temperature. Intermediate microwave power between 3 and 4 W/g, the lowest tested pressure level of 7 mbar and low product temperatures of 30–35 °C, resulted in the highest survival and activity of the bacterial cells. With these results, it could be shown that microwave-vacuum drying appears as a promising alternative drying technique for the preservation of starter and probiotic cultures.     

ACCELERATED STORAGE TESTING OF FREEZE-DRIED IMMOBILIZED LACTOBACILLUS ACIDOPHILUS-FERMENTED BANANA MEDIA

ABSTRACT

Freeze‐drying was applied as a drying method for the production of dehydrated immobilized Lactobacillus acidophilus‐fermented banana medium containing high levels of probiotics and appropriate prebiotic (mainly fructooligosaccharides) contents in an attempt to develop dried synbiotic products. Ca‐alginate and κ‐carrageenan were used for the immobilization of L. acidophilus, and the immobilized bacteria were employed directly in the banana media fermentation. The fermented products were then freeze‐dried. Results indicated that cell immobilization could provide effective protection to L. acidophilus, and reduce the damage caused by freezing and freeze‐drying. Meanwhile, accelerated storage testing using temperatures of 50, 60 and 70C was applied to the dehydrated products. Gel‐entrapped L. acidophilus appeared to have lower decimal reduction, and Ca‐alginate immobilized cells had a better survival than κ‐carrageenan. Results of accelerated storage test showed that immobilization could effectively increase the thermal resistance of entrapped microorganisms, and Ca‐alginate showed a better effect than κ‐carrageenan, and the beneficial effects increased with the decrease of the storage temperature. The freeze‐dried products exhibited little hygroscopicity because of the consumption of monosaccharides during fermentation. Results revealed that freeze‐drying could be used as a proper method for the development of dried product of immobilized L. acidophilus‐fermented banana media, and accelerated storage test could be used to evaluate the storage stability of the dried products.

PRACTICAL APPLICATIONS

This research demonstrates the feasibility of the application of accelerated storage testing to the prediction of shelf life and storage stability of freeze‐dried probiotic products manufactured by lactic acid bacteria fermentation. The Arrhenius equation was associated in this study. This will provide an efficient approach for the estimation of shelf life of probiotic products.

     

Study of the effects of spray‐drying on the functionality of probiotic lactobacilli

Three probiotic lactobacilli strains were spray‐dried in 20% (w/v) skim milk and submitted to a simulated gastrointestinal digestion. Fresh or spray‐dried cultures were administered to mice for 5 and 10 days, and Immunoglobulin A (IgA)‐producing cells were enumerated in the small intestine by immunohistochemistry. Spray‐drying significantly enhanced the resistance of Lactobacillus paracasei A13 and Lactobacillus casei Nad to a simulated gastrointestinal digestion (0.96 and 1.95 log orders, respectively), compared with fresh cultures. Also, a significant higher number of IgA‐producing cells were induced by spray‐dried cultures compared with fresh cultures. Spray‐drying is a suitable, but strain‐dependent, technological process for the development of probiotic cultures in skim milk with increased functionality.     

How probiotics face food stress: They get by with a little help

Abstract

The dietary consumption of probiotics in the form of pharmaceuticals or functional food can improve human health and contribute to disease prevention. However, the biological activity and health potential of food-delivered probiotics can be severely compromised by the stress conditions encountered by the microorganisms throughout the manufacture process, from probiotic preparation to their inclusion into food, subsequent storage and ingestion. Here, we give an account of the stress factors that can have major negative impacts on probiotic viability and functionality, with a focus on food-related environmental adverse conditions. We also describe some of the mechanisms elicited by the microbial cells to counteract these stresses and summarize a few relevant approaches proposed in literature to develop more robust and competitive probiotics by enhancing their stress tolerance, with the aim to improve the efficacy and health value of probiotic functional food.     

Pulsed electric field pre-treatment for enhanced bacterial survival after drying: Effect of carrier matrix and strain variability

Previous studies suggest that increased intracellular trehalose concentrations can result in increased robustness of probiotics and starter cultures after processing. We evaluated whether pulsed electric field (PEF) pre-treatment, resulting in increased intracellular trehalose concentrations, can lead to enhanced survival during spray drying and subsequent storage and what the effect of the carrier matrix during drying herein is on survival. The applied PEF pre-treatment resulted in increased survival of Lactobacillus plantarum WCFS1 after spray drying and subsequent storage in reconstituted skim milk (RSM), though not in the other evaluated carrier matrices. The same PEF pre-treatment in lactose instead of trehalose electroporation medium resulted also in an increased survival after spray drying in RSM. Further analysis of intracellular trehalose and lactose concentrations revealed that survival after spray drying cannot only be explained by intracellular trehalose and/or lactose concentrations. Experiments with other bacterial strains indicated that strain variability cannot be neglected when designing a process aimed at enhanced bacterial survival after processing. Overall, PEF pre-treatment is promising for enhancing survival of bacteria after drying and storage, though further understanding of the applicability is required for industrial application.Industrial relevance textProbiotics and starter cultures are very common in food industry. Survival of these cultures during drying processes is essential for their application. A pulsed electric field pre-treatment resulting in increased bacterial survival after spray drying and powder storage may contribute to more energy efficient production processes of dried bacterial cultures.     

Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG

The objective of this work was to evaluate the applicability of spray drying in the production of skim milk-based preparations containing probiotic bacteria Lactobacillus rhamnosus GG (ATCC 53103). Furthermore, oligofructose-based or polydextrose-based prebiotic substances were also included in the carrier matrix to assess their protection capacity. When reconstituted, skim milk was used as a spray drying carrier; a microbial survival rate of 60% was achieved at an outlet temperature of 80 °C. Partial substitution of the solids content of the reconstituted skim milk by prebiotic substances also resulted in a high level of survival. However, the storage stability of the dried powder was reduced as the amount of skim milk solids in the carrier was lowered. The spray drying media were further calorimetrically characterized in terms of their glass transition temperatures so as to evaluate the contribution of glassy state on the maintenance of bacterial survival during storage. Although all evaluated carriers were in the glassy state, differences were observed in their capacity to confer protection on the probiotic bacteria. Flow cytometric assessment in combination with functional dyes was applied as a diagnostic tool to evaluate the type of cellular injuries which occurred upon spray-drying. Cell death was caused mainly by damage to cell membranes and the degree of membrane disintegration increased progressively as the outlet temperatures increased.     

Pediococcus acidilactici as a potential probiotic to be used in food industry

The objective of this study was to compare the results obtained between a commercial probiotic bacterium and a strain of Pediococcus acidilactici isolated from a food matrix. Besides the characterisation of the isolates, the main focus of this work was to evaluate their ability to survive through simulated gastrointestinal tract passage as well as the effect of the spray drying process to be used as concentrated cultures in the food industry. Both lactic acid bacteria produced any of the virulence determinants investigated; they exhibited no significant antibiotic resistances and <1 log unit reduction in viable counts was obtained after exposure to simulated gastrointestinal tract conditions. Concentrated cultures of P. acidilactici HA‐6111‐2 in 10% (w/v) of reconstituted skim milk were obtained by spray drying and, after 60 days of storage at 4 °C, <1 log unit reduction in viable count was recorded. In conclusion, from these preliminary characterisation tests and its extended viability in the dried form, this P. acidilactici strain should be considered as a potentially useful probiotic.     

Effects of conventional and nonconventional drying on the stability of Bifidobacterium animalis subsp. lactis INL1

Freeze, spray, vacuum, microwave vacuum‐ and microwave freeze‐drying were applied to Bifidobacterium animalis subsp. lactis INL1. Freeze and microwave freeze‐drying showed the highest survival after drying. When a storage test (25 °C; oxygen) was performed, these cultures were the most sensitive ones at a_w = 0.23, but the addition of lactose improved their stability after 8 weeks. Flow cytometry was useful to assess viability after drying but not during storage. Our results show that dehydration technologies other than freeze‐drying might be suitable alternatives that deserve further investigation for the preservation of sensitive probiotic bacteria. Microwave drying rendered cultures of comparable characteristics to their conventional counterparts, requiring significantly shorter drying times.     

Protecting probiotic bacteria by microencapsulation: challenges for industrial applications

The use of probiotic bacteria in novel foods to provide beneficial health effects is today of increasing interest in the food industry. The process stability of probiotics is, however, not always optimal. Microencapsulation technology can be used to maintain the viability of probiotic bacteria during food product processing and storage. Both true microcapsules with coating as well as microspheres where the bacteria are evenly spread in the coating material are discussed. It is important that encapsulation keeps the probiotics active through the gastrointestinal tract and releases them in their target organ. The survival of microencapsulated cells in simulated gastric conditions is therefore also reviewed. Polysaccharides like alginate, gellan, κ-carrageenan and starch are the most commonly used materials in microencapsulation of bifidobacteria and lactobacilli. Techniques commonly applied for probiotic microencapsulation are emulsion, extrusion, spray drying, and adhesion to starch. Bead stability can be improved by using different coating materials, e.g. chitosan. Future challenges in the field include recognition of new potent applications, selection of appropriate techniques, materials and bacterial strains, and minimizing the extra costs incurred by microencapsulation.     

Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions

This study investigated the stability of freeze dried and fluid bed dried alginate microcapsules coated with chitosan containing model probiotic bacteria, Lactobacillus plantarum, during storage for up to 45 days at different water activities (0.11, 0.23, 0.40 and 0.70) and temperatures (4, 30 and 37 °C). The loss in cell viability was around 0.8 log in the case of fluid bed drying and around 1.3 in the case of freeze drying, with the former method resulting in dried capsules of smaller size (~ 1 mm vs 1.3 mm), more irregular shape, and with a rougher surface. In both cases, the water activity and water content were less than 0.25 and 10% w/w, respectively, which favours high storage stability. The storage stability studies demonstrated that as the water activity and temperature decreased the survival of the dried encapsulated cells increased. Considerably better survival was observed for fluid bed dried encapsulated cells compared to freeze dried encapsulated cells and freeze dried free cells with 10% sucrose (control), and in some cases, e.g. at 4 and 30 °C at water activities of 0.11, 0.23 and 0.40, there was more than 1 log difference after 45 days, with concentrations higher than 10⁸ CFU/g after 45 days of storage. The results indicate that fluid bed drying is an effective and efficient manufacturing method to produce probiotic containing capsules with enhanced storage stability.     

Influence of spray drying conditions on functionality of dried whole egg

Instead of traditional liquid eggs, more and more dried whole egg products are being used in the manufacture of fine bakery goods, since dried semi‐finished products have some advantages regarding storage, handling and microbiological safety. However, the spray drying process leads to changes in sensitive egg components, eg proteins, and affects the functional properties of whole egg after reconstitution. Knowledge of the influence of drying conditions on the resulting functionality is necessary to enable the production of dried egg with tailor‐made properties for applications in different food products. Some of these relationships were determined using a pilot spray dryer for drying experiments and an appropriate statistical evaluation of the results. It could be demonstrated that higher temperatures during spray drying lead to a considerable decrease in the foaming properties of whole egg powder but increase the capability of emulsion stabilisation. Additionally, a simplified example demonstrates the use of these relationships for the selection of optimal drying conditions of whole egg with respect to its application in a cake with higher fat content. © 2002 Society of Chemical Industry