Various supports and immobilization/encapsulation techniques have been proposed and tested for application in functional food production. In the present review, the use of probiotic microorganisms for the production of novel foods is discussed, while the benefits and criteria of using probiotic cultures are analyzed. Subsequently, immobilization/encapsulation applications in the food industry aiming at the prolongation of cell viability are described together with an evaluation of their potential future impact, which is also highlighted and assessed.
Probiotics have rapidly gained interest in the area of self-care and complementary medicine under the general term “functional foods.” Modern consumers are increasingly interested in their personal health and particularly in foods which are capable of preventing and/or curing illness. Microbes have been used for years in food and alcoholic fermentations but only recently have undergone scientific scrutiny to examine their possible health benefits.
The word “probiotic” comes from the Greek words “pro” and “biotic,” meaning “for the life.” The concept of “probiotics” appeared a long time ago. The Nobel laureate Elie Metchnikoff was the first microbiologist in the beginning of the 20th century who suggested that the longevity of Bulgarian peasants could be related to their large consumption of sour milk containing
A variety of microorganisms have been studied for potential probiotic effects. Most microbial strains with probiotic activity belong primarily to
Most common microorganisms studied for probiotic properties.
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Bifidobacteria | |
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Other bacteria | |
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Yeast | |
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Immobilization/encapsulation of probiotics is an exciting field of food technology that has emerged and developed rapidly in the past decade. The most excellent application of probiotic immobilization technology is the controlled and continuous delivery of cells in the gut. The potential benefit of this therapeutic strategy is to maintain greater cell viability despite the acidity of the stomach. In their viable state, probiotics exert a health benefit on the host.
There is growing scientific evidence to support the concept that the maintenance of healthy gut microbiota may provide protection against gastrointestinal disorders, such as gastrointestinal infections and inflammatory bowel diseases [
Most important beneficial effects of probiotics.
Beneficial effect | Probiotic microorganism | Type of trial | Outcome | Reference | |
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Metabolism | Lactose digestion |
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Target group of patients, lactose maldigestion |
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Lipid metabolism |
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Randomized |
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Oxalate metabolism |
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Target group of patients, stone-forming patients |
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Chronic intestinal inflammatory and functional disorders | Inflammatory bowel diseases (IBD): Crohn’s disease |
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Pilot study |
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Ulcerative colitis |
Combined lactobacilli and enterococci | Randomized | Effective treatment by mediating immunological response | [ | |
Irritable bowel syndrome (IBS) |
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Double blind, placebo controlled, and parallel designed |
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Allergic diseases | Eczema |
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Randomized, double blind, placebo controlled |
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Atopic dermatitis |
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Randomized, double blind, placebo controlled |
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Allergic rhinitis |
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Randomized, double blind controlled |
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Asthma |
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Randomized, placebo controlled |
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Reduction of risk factors of infection | Infectious diarrhea |
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Randomized, single blind | Synbiotic mixture showed reduction in diarrhea duration | [ |
Necrotizing enterocolitis (infants) |
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Randomized, placebo controlled | Protective effect in clinical NEC | [ | |
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Randomized, double blind, placebo controlled | Positive effect on the eradication of |
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Respiratory tract infections | Ear, nose, and throat infections |
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Randomized, double blind, and placebo controlled |
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Malignancy | Cervical cancer |
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Prospective controlled pilot study | Probiotic studies promoted the clearance of HPV-related cytological abnormalities | [ |
The overall objective of this review is to analyze and assess the data on immobilization technology of probiotic microorganisms for application in food production.
Several aspects, including safety, functional and technological characteristics, have to be taken into consideration in the selection process of probiotic microorganisms. Many microorganisms could be considered as potential probiotics, but only a few are able to satisfy the necessary criteria.
Safety aspects include specifications such as origin (healthy human gastrointestinal tract), nonpathogenicity, nondigestive upsets, and nonantibiotic resistance characteristics.
Functional aspects include viability and persistence in the gastro-intestinal (GI) tract, surviving the digestive stresses [
Careful screening of probiotic strains for their technological suitability can also allow selection of strains with the best manufacturing and food technology characteristics. Moreover, they should not produce off-flavours [
An overview of the most significant criteria to define a probiotic microorganism is presented in Table
Criteria used to define a probiotic microorganism.
Safety criteria | Be of human origin |
Nonpathogenic in nature | |
Generally recognized as safe (GRAS) | |
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Functional criteria | Be resistant to destruction by gastric acid and bile salts |
Adhere to intestinal epithelial tissue | |
Be able to colonize the gastrointestinal tract, even in the short term | |
Modulate immune responses | |
Produce antimicrobial substances | |
Influence human metabolic activities (i.e., cholesterol assimilation, lactase activity, vitamin production, etc.) | |
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Technological criteria | Be resistant to destruction by technical processing |
Be subjected to scale-up processes |
It is essential that probiotics must be considered as “generally recognized as safe” (GRAS) organisms for human use according to the US Food and Drug Administration [ it is highly recommended that strains used for products addressed to humans should be of human origin. Additionally, a probiotic strain is expected to function better in a similar environment from where it was originally isolated (e.g., human GI-tract). Generally, probiotics should be isolated from healthy human GI tract. It is also considered that the safety criteria depend on our experience in food fermentations; there should be no association with disease. Most intestinal microorganisms are not considered pathogenic in healthy individuals, but some intestinal bacteria are potentially pathogenic. Their growth and metabolism are influenced by the normal immune system in the digestive tract. The pathogenic microbes can potentially cause an infection even in a healthy host; metabolic activity in the food matrix and in the intestine following consumption is an important safety criterion. For example, although tolerance of bile salts is an essential criterion for the selection of potential probiotic strains, microbial bile salts hydrolase activity has been mooted to be potentially detrimental to the human host, and thus it is yet not completely clear whether it is in fact a desirable trait in a probiotic bacterium [ the selected strains should not carry transmissible antibiotic resistance genes.
For the selection of a probiotic strain, several criteria of functionality have to be considered. The functional criteria of probiotics should be established based on both
The survival of different probiotic strains in different parts of the GI-tract may greatly vary. Some strains are rapidly killed in the stomach, while others are able to pass through the whole gut in high numbers [
The reduction of viable cell levels might not always constitute a major issue, as a high number of studies reporting that nonviable probiotics could also have beneficial effects on human health or even be more efficient than alive cells are available [
On the other hand, maintenance of cell viability is an essential requirement for the prevention and/or treatment of many disorders; that is, a daily dose of at least 108 cells was required to restore and maintain a normal urogenital flora in women [
The health benefits of potential probiotic strains should be also assessed. Potential benefits may vary from maintenance of normal intestinal flora [
Even though a probiotic strain fulfills the necessary safety and functional criteria, its selection should also satisfy technological criteria, as aspects related to probiotic food production and processing are also very important.
Viability of bacteria is often reduced during the food manufacture, distribution, and storage. Non-viable cultured products usually have longer shelf-life and easier storage which favour the adoption of the technology by the industrial sector, but it has been claimed that only probiotic products with viable microorganisms have beneficial health effects.
As it is strongly suggested that probiotic products should contain an adequate amount of live bacteria (at least
Many surveys have shown large fluctuations and poor viability of probiotic bacteria, and especially bifidobacteria, in food products, such as yoghurt preparations [
As with all fermented dairy products containing living bacteria, products containing bifidobacteria must be cooled during storage, which is necessary both to guarantee high survival rates and to ensure product stability [
The terms immobilization and encapsulation have been used interchangeably. Immobilization refers to the trapping of a material within or throughout a matrix, while encapsulation is the process of forming a continuous coating around an inner matrix that is wholly contained within the capsule wall as a core of encapsulated material. In both cases, the bidirectional diffusion of molecules, such as the influx of oxygen, nutrients, and growth factors, essential for cell metabolism and the outward diffusion of waste products should be permitted.
Immobilization techniques often mimic nature, as naturally many microorganisms own the ability to adhere to and survive on different kinds of surfaces, and thus cells may grow within natural structures.
The immobilization methods can be divided into the following four major categories based on the physical mechanism employed (Figure entrapment within a porous matrix due to cells penetration until their mobility is obstructed by the presence of other cells or to formation of porous material attachment or adsorption on solid carrier surfaces by physical adsorption due to electrostatic forces or by covalent binding between the cell membrane and the carrier, self-aggregation by flocculation (natural) or with artificially induced cross-linking agents, mechanical containment behind a barrier which could be either a microporous membrane or a microcapsule.
Basic methods of cell immobilization [
However, not all carriers are suitable for food production. Material used as a carrier should (a) have chemical, physical, and biological stability during processing and in the reaction conditions, (b) have sufficient mechanical strength, especially for its utilization in reactors and industry, (c) be nontoxic both for the immobilized cell and for the product, and (d) have high loading capacity. Material availability and cost-effectiveness of the immobilization process always have to be considered. Other criteria, such as physical characteristics (porosity, swelling, compression, and mean particle behavior), as well as possibility for microbial growth, biodegradability, and solubility, are application specific and should be also taken into account. Table
Prerequisites of immobilization supports and advantages of cell immobilization.
Prerequisites of immobilization supports | Advantages of cell immobilization |
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Recent examples of research and applications on cell immobilization have emerged a series of advantages, which are summarized in Table
Foods used for dissemination of probiotics are usually fermented foods even if probiotics could also be present in infant formulas, fruit drinks, whey drinks, and sweet milk. Fermented milk and cheese are the most common foods containing probiotics [
In Europe, probiotic applications are restricted to fermented milk products. In the United States, however, probiotics are found most frequently in the supplements sector and in yoghurts, while in Japan and Korea the use of probiotics is also widespread in food products that claim to assist the digestion process. On the other hand, research is also currently oriented to nondairy foods, like fermented meat and bakery products.
Among the numerous immobilization supports, only a few are considered suitable for food production. For example, inorganic materials are usually excluded because they are characterized as unsuitable for human or animal nutrition. Instead, biopolymers and natural supports of food-grade purity are preferable. It is also very interesting to exploit materials with nondigestible carbohydrates and to investigate their application in probiotic food production.
Table
Characteristic examples of application of probiotic cell immobilization in food production.
Immobilization/encapsulation support | Probiotic microorganism | Probiotic food product | Reference |
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Alginate encapsulation |
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Mayonnaise | [ |
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Apple pieces, quince pieces |
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Fermented milk | [ |
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Apple, pear pieces |
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Cheese | [ |
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Chitosan coated alginate beads |
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Yogurt | [ |
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Fibres |
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Apple juice, chocolate coated breakfast cereals | [ |
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Calcium induced, encapsulated alginate starch |
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Yogurt | [ |
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Microencapsulation in alginate |
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Sausages | [ |
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Whey protein |
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Biscuits, frozen cranberry juice, and vegetable juice | [ |
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Calcium alginate |
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Tomato juice | [ |
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Rinds of durian, mangosteen, and jackfruit |
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Soy milk | [ |
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Calcium alginate |
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Yogurt | [ |
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Sodium alginate |
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Synbiotic milk chocolate | [ |
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Fruits, oat pieces |
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Yogurt | [ |
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Wheat grains |
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Fermented sausage | [unpublished results] |
Microencapsulation has been reported as a technology that can provide protection to sensitive cultures from high oxygen levels [
Alginates are naturally derived linear copolymers of 1,4-linked
Calcium alginate microspheres can be produced by both extrusion and emulsion techniques [
Extrusion is the oldest and the most common approach to make capsules with hydrocolloids and might be achieved by simply dropping an aqueous solution of probiotics into a gelling bath. The size and shape of the beads usually range 2–5 mm and depend on the diameter of the needle and the distance of free fall [
In the emulsion technique, a small volume of cell-polymer suspension is added to a large volume of oil, and the mixture is homogenized to form a water-in-oil emulsion. Very often, the pH is reduced by addition of an oil-soluble acid, for example, acetic acid, enabling initiation of gelation with Ca2+. The size of beads depends on the speed of agitation and the type of emulsifier used. Therefore, it enables the production of the targeted microcapsules size [
Figure
Steps of extrusion and emulsion processes and the chemical structure of the alginate residues along with a schematic diagram of the microorganisms and the hydrogels.
An important challenge for probiotic encapsulation is to reduce the particle size, because it can negatively affect the textural and the sensorial properties of the product. For example, consumers detected a grainy texture in yogurts containing encapsulated bifidobacteria (size range particles about 22–50
Another issue that should be addressed is that the presence of residual oil on capsule surface produced by emulsification is detrimental to the texture and the organoleptic properties of the product. Also, capsules incorporation in diet products is hampered, and the residual oil, surfactant, or emulsifier can be toxic for probiotic cells.
The survival of the microencapsulated probiotics
Microencapsulation also appeared to create anoxic regions inside the microcapsules, therefore reducing oxygen, which prevented viability losses of oxygen-sensitive probiotic strains, in addition to protecting the cells against the acid conditions in yoghurt [
Ca-alginate entrapment of
Similarly, survival of calcium-induced alginate-starch encapsulated
A symbiotic pastry product was previously prepared by incorporating free or encapsulated
On the contrary, encapsulation of
In another study, it was reported that microencapsulation in alginates resulted in enhanced resistance of
Encapsulation of probiotics in whey protein gel particles may offer protection during food processing and storage. The protein microcapsules containing the encapsulated bacteria can be produced by emulsion and spray drying or extrusion and freeze drying and they may be incorporated in various products, such as yoghurt, cheese, and biscuits, to confer probiotic properties [
The efficacy of whey protein isolate as an encapsulation matrix for the maintenance of
Accordingly, cell immobilization of the same strain (
Direct dispersion of fresh cells in a heat-treated whey protein suspension followed by spray drying was also proposed as an alternative and less destructive microencapsulation method, with survival rates after spray drying of 26% for
Likewise, the effect of whey protein isolate gel microentrapment on the viability of
Fruits contain non-digestible carbohydrates, which constitute the base for cell immobilization. Apple and quince pieces proved to be suitable supports for immobilization of
Fruit and oat pieces were also recently proposed as vehicles for delivery of
Attempts were carried out to combine the beneficial effects of probiotics with fruit and vegetables by applying the vacuum impregnation process. It was shown that it is possible to introduce microbial cells into structural matrix of fresh apple tissue by using impregnation liquid inoculated with
The aim of preservation techniques for foods of concern in “Ibero-America” (CYTED Program), carried out from 1999 to 2004, was to analyze the feasibility of atmospheric and/or vacuum impregnation treatments in order to incorporate physiologically active compounds into plant tissues without destroying the initial food matrix. The above research contributed significantly in the development of functional fruit and vegetable matrices enriched with probiotics [
Cereals, which also contain non-digestible carbohydrates, could be applied as supports for cell immobilization. During the last years, several encapsulation techniques using cereal fractions have been tested in order to improve the viability of the probiotic strains in functional foods [
Based on the above perspective, wheat dextrin, polydextrose, apple fibre, and inulin were considered promising carriers of
Recently, production of probiotic dry fermented sausages containing immobilized
Finally, efforts to immobilize probiotic strain on agricultural wastes were recently carried out. Rinds of durian, mangosteen, and jackfruit were used as supports for immobilizing strains of
Despite the plethora of probiotic products and the immobilization supports proposed by several researchers, the immobilized cell technology has not yet been widely adopted by the industrial sector, mainly due to safety issues related to the immobilization agents, confirmation of the stability, and functionality of bioactive cultures and the lack of processes that can be readily scaledup. Ongoing research aims at resolving the above issues, as immobilization is a successful way of protecting and improving cell viability. The assessment of the industrial feasibility of immobilization technology is mandatory for providing cost-effective, large-scale quantities of probiotic products for specific clinical and/or commercial use.
The crucial factors for the implementation on an industrial level are carrier materials, immobilization technology, and bioreactor design. Although research on immobilized cells has been carried out for several years, many difficulties related to the application at industrial scale still exist. The two most important disadvantages that should be always kept on mind are complexity of production process and cost limitations. At the moment, the major challenges for successful application of immobilized cell technology in industrial probiotic food processing are evidence of enhancement of cell viability, effective clinical outcome versus free cells and finetuning of the organoleptic characteristics. In fact, engineering problems linked to choice of the carrier and reactor design are complicated by the safety of immobilizing agents and the effects of immobilization on the sensory attributes of the final products. Future research should be focused on overcoming the gap between conditions at research level and demands for large-scale applications, improving existing manufacturing technologies, and choosing new processing conditions and new carrier materials. Furthermore, future studies should be oriented to preservation and storage techniques that could be easily adopted at the industrial level.
For successful immobilization and cultivation of probiotic cells, the immobilization material must be conducive to cell viability and function (biocompatible) within specific food systems. Hence, immobilization supports of food grade purity, such as natural supports, are considered advantageous for food production. Microcapsule or bead systems using various biopolymers are very easy to prepare on a lab scale. However, the scaling up of the process is very difficult, and processing costs are very high. In addition, mechanical instability is an important disadvantage of gels. It has been often noticed that the gel structure is being destroyed due to cell growth and intensive carbon dioxide production.
In the near future, multiple deliveries are expected to be the key factor, and thus a new area of complex nutritional matrices will be augmented. For example, coencapsulation of probiotic cultures with certain food ingredients may be beneficial, as at the same time it enables introduction of bioactive compounds, while the positive effects of probiotics can be enhanced with the right selection of substances. Hence, coimmobilization of probiotic microorganism with prebiotics, antioxidants, peptides, or immune-enhancing compounds is becoming especially attractive in future perspectives.
A number of efficient materials and the associated controlled release mechanisms are currently under investigation. It is expected that new innovative ways of administration and delivering of probiotics will be developed shortly. However, more research is still required for the selection of immobilization supports that can trigger successful adhesion to specific intestinal cells, therefore achieving targeted delivery of probiotic bacteria to various sites within the GI tract. More
Additionally, new food regulation should specify labelling including the strain and the number of viable cells at the end of the shelf-life of probiotic-claimed foods. Such directives are considered crucial for the development of industrial and commercial consciousness and for the consumer protection.
Finally, the development of novel functional foods is a major challenge to address the expectation of consumers for healthy and beneficial food products. Industries should overcome the possible difficulties and find ways to exploit the advantages offered by the immobilized cell technology with an adequate cost. It is evident that the probiotic market has a strong future, as the benefits provided by probiotics consumption are now well documented, and thus consumer requirements are expected to increase.