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The natural inhabitants of the gastrointestinal tract play a key role in the maintenance of human health. Over the last century, the changes on the behavior of our modern society have impacted the diversity of this gut microbiome. Among the strategies to restore gut microbial homeostasis, the use of probiotics has received a lot of attention. Probiotics are living microorganisms that promote the host health when administered in adequate amounts. Its popularity increase in the marketplace in the last decade draws the interest of scientists in finding suitable methods capable of delivering adequate amounts of viable cells into the gastrointestinal tract. Encapsulation comes into the scene as an approach to enhance the cells survival during processing, storage and consumption. This paper provides a comprehensive perspective of the probiotic field at present time focusing on the academia and industry scenarios in the past few years in terms of encapsulation technologies employed and research insights including patents. The analysis of the encapsulation technologies considering food processing costs and payload of viable bacteria reaching the gastrointestinal tract would result into successful market novelties. There is yet a necessity to bridge the gap between academia and industry.
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AIMS Materials Science, 3(1): 114-136.
DOI: 10.3934/matersci.2016.1.114
Received: 25 September 2015
Accepted: 11 January 2016
Published: 17 January 2016
Encapsulation of probiotics: insights into academic and industrial
Fernanda B Haffner, Roudayna Diab *, and Andreea Pasc
SRSMC UMR 7565, Université de Lorraine, CNRS, Bd des Aiguillettes-BP 70239 F-54506
Vandœuvre-lès-Nancy, France
* Correspondance : E-mail:; Tel: (+33)-03-83-68-46-61.
Abstract: The natural inhabitants of the gastrointestinal tract play a key role in the maintenance of
human health. Over the last century, the changes on the behavior of our modern society have
impacted the diversity of this gut microbiome. Among the strategies to restore gut microbial
homeostasis, the use of probiotics has received a lot of attention. Probiotics are living
microorganisms that promote the host health when administered in adequate amounts. Its popularity
increase in the marketplace in the last decade draws the interest of scientists in finding suitable
methods capable of delivering adequate amounts of viable cells into the gastrointestinal tract.
Encapsulation comes into the scene as an approach to enhance the cells survival during processing,
storage and consumption.
This paper provides a comprehensive perspective of the probiotic field at present time focusing
on the academia and industry scenarios in the past few years in terms of encapsulation technologies
employed and research insights including patents. The analysis of the encapsulation technologies
considering food processing costs and payload of viable bacteria reaching the gastrointestinal tract
would result into successful market novelties. There is yet a necessity to bridge the gap between
academia and industry.
Keywords: bacteria; microencapsulation; microparticles; probiotics; process
GIT: gastrointestinal tract, CFU: colony-forming unit, US FDA: United States Food and Drug
Administration, MRS: Man, Rogosa and Sharpe
AIMS Materials Science Volume 3, Issue 1, 114-136.
1. Introduction
Probiotics are living microorganisms that promote the host health when administered in
adequate amounts [1]. Commonly the scientific community accepts a daily consumption of 108 to
109 CFU/g (Colony Forming Units per gram) of probiotic viable cells as being the minimum amount
required to confer the desired benefits [2]. Thus US FDA recommends a minimum concentration of
106 CFU per ml or per g of probiotic viable cells right at consumption in the food product [3].
However, to date there are no uniform therapeutic dosing recommendations for probiotic intake.
Doses range from 107 CFU /day to 1012 CFU/day, depending on the clinical endpoint, the probiotic
genus, species or strain [4].
The Russian Nobel Prize Eli Metchnikoff was the first one to suggest the beneficial role of the
gastrointestinal bacteria at the beginning of the twentieth-century. He based his hypothesis on the
observation of the longevity of Bulgarian peasants and their frequent intake of fermented dairy
products. Metchnikoff postulated that the lactic acid bacteria replaced the pathogenic
microorganisms in the guts and the result was a decrease in certain illnesses [1]. At the same period
of time, a French pediatrician called Henry Tissier observed the low numbers of a peculiar Y-shaped
bacterium in the stools of infants facing diarrhea. He observed that healthy subjects had plenty of
these ‘bifid’ microorganisms, which led him to suggest its administration to restore the microbiota of
the ill children. Metchnikoff and Tissier were thus the pioneers of the probiotic field, although the
term probiotic was not utilized until decades later [1].
Under natural circumstances humans should not need probiotic supplements with the exception
of the public holding any type of special condition, e.g. long-term use of antibiotics. However,
changes of the behaviors of our society challenge the inhabitants of the gut [5]. Sterile food
consumption, increased hygienization, stress and antibiotics administration are some of the
antagonists of our commensal microbiota, which can be potentially normalized by the ingestion of
Today, a wide range of probiotic products, mainly dairy, is in the market, and more are yet to
come, especially non-dairy novelties [6]. The low-cholesterol diets, lactose intolerance and milk
protein allergy public, vegans and vegetarians are some of the future potential customers for these
products. Herein some examples: table olives [7]; apple juice [8]; goat’s milk ice cream [9]; green
tea [10]; kefir [11]; dry apple snack [12]; orange, pineapple and cranberry juices [13]; pomegranate
and cranberry juices [14]; carrot and watermelon juice [15]; fresh apple wedges [16,17];
chocolate [18] and cereals [19,20]. Some probiotic products have the drug status and are prescribed
as anti-diarrheic, e.g., Lacteol®, Lactobiane® and Bacilor®. The global economic scenario of
probiotics is very encouraging. Probiotic ingredients and supplements reached approximately $23.1
billion in 2012; and the growth forecast is in the $36.7 billion range to 2018, with a compound
annual growth rate of 6.2% in the next four years [21].
The maintenance of the cell viability in probiotic-containing products is still considerably
challenging. The microorganisms must survive during the industrial processing, which is often
detrimental to the cell viability in the final product. As these products are intended for oral use, the
remaining viable cells undergo further stresses during the gastrointestinal passage due to changes in
pH or the contact with bile salts, which might disrupt the membrane of microorganisms. As a
consequence, additional loss in cell viability is expected before they even reach their target.
AIMS Materials Science Volume 3, Issue 1, 114-136.
In this context, the encapsulation of probiotics could provide the required protection to the
administrated bacterial cells and thus, increase the delivered amount. Piano et al. demonstrated
through several clinical studies that microencapsulated probiotics were significantly more effective
to deliver viable cells to the colon than the non-encapsulated counterparts [22,23]. The authors
reported that subjects who received microencapsulated cells showed a similar intestinal colonization
profile with respect to the subjects who received 5-time higher amounts of non-encapsulated cells.
In order to successfully encapsulate viable cells into foodstuff, it is of paramount importance to
preserve the bacterial viability in all of the manipulation procedures along with choosing accordingly
the encapsulation materials, which must be food compatible [24,25]. Additionally, the microcapsules
must maintain the cell viability during their storage alone or when added to food carriers. For
instance, high relative humidity in the final product will favor the oxidative process of the matrix and
therefore be detrimental to the cells [26]. Teixeira et al. reported an optimal relative humidity of 7
11% for bacterial survival [27].
Although valuable and encouraging research has been published over the years and
commercialization of products is being made, the information exchange between industry and
academia should noticeably intensify. Encapsulation of probiotics and food delivery systems are a
rapid growing research theme. This paper aims to give an overview of the vast probiotic field on
what is being done in the academia and industry scenarios in the past few years, in terms of
technologies employed and research insights. For such, mostly recent and/or other important
published work including patents is the focus of this review.
2. Microcapsules Morphology and Encapsulating Agents
Scientists assess a multitude of technological approaches for obtaining microcapsules. The final
morphology obtained and diameter will depend on technological process and materials implied
during the microcapsule formation.
Several materials have been extensively researched up to date for probiotic encapsulation
purposes. Some are very popular with a long reputation of use such as alginate. Alginate forms
matrix microspheres that can be obtained by extrusion. When a hydrophobic coating is applied over
the alginate matrix, the result is a shell-matrix microcapsule, and if the matrix is a bulk, then the
microcapsule is a shell-core-type (Figure 1) [28,29]. Materials used for probiotic encapsulation were
previously reported in several reviews [30–33].
Table 1 resumes pros and cons for each technology correlating them with current encapsulation
agents and final capsules sizes. To access information on cell immobilization procedures and more
details on materials used for microencapsulation refer to Poncelet et al. [34] and Oxley et al. [28],
respectively. In terms of technological processes spray drying, spray cooling, spray coating,
emulsification, liposomes, coacervation and extrusion are addressed in more detail. It is important to
keep in mind that frequently a combination of these technologies is applied in order to obtain the
intended encapsulated material.
AIMS Materials Science Volume 3, Issue 1, 114-136.
Figure 1. Fundamental components of an encapsulated particle: Matrix microsphere,
shell-matrix and shell-core microcapsules.
Table 1. Correlation of encapsulation technologies with capsules sizes, most current
encapsulation agents, pros and cons.
Most current
Pros Cons
Spray drying 5 to 150
Inulin [36,37],
acacia gum [38],
locust bean gum
[39], starch [8],
soy protein [38],
whey protein
skim milk [36],
chitosan [42]
[43], alginate [43]
1. Mass production,
2. Continuous process,
3. Material
4. Well established in the
food industry for other
5. Inexpensive.
1.Loss of cell viability due to high
2.Mostly used with aqueous suspensions,
i.e. shell material should be soluble in
water [28].
Spray cooling 20 to 200
Cocoa butter [44],
Palm oil [45],
Palm kernel oil
1. Mass production,
2. Continuous process,
3. The least expensive
technique [46];
4. Mild temperatures
1.Lower load (10-20%) when compared
to spray drying (5-50%) [35].
2.Encapsulated ingredient may be on the
surface and in contact with environment
(matrix spheres) [46],
3.Difficulty to delay the release of water-
soluble ingredient over 30min [46].
Freeze drying -
Starch [47], soy
protein [20,48],
casein [48], whey
protein [48], skim
milk [48]
1.Excellet final dried
material suitable for most
food applications [28],
2.Suitable for sensitive
materials as probiotics.
1.High costs,
2. Cell damage with eventual crystal
formation, if not done correctly [28],
3.Eventual need of cryoprotectants.
Spray coating 5 µm to 1
Shellac [39],
casein [49]
1.Control release with
addition of different
2.Increase in storage
1. May introduce forces that can damage
the cells [28],
2. Although temperature is lower, the
exposure could be longer along with
oxygen exposure [28].
200 nm
to 1 mm
Inulin [50], K-
carrageenan [51],
alginate [52]
1.Easy technique,
2.Usual high survival rate
of bacteria.
1.Costly to scale-up,
2.Polydispersity of material,
3.Shape variation of material,
4.Prolonged shear forces may cause
AIMS Materials Science Volume 3, Issue 1, 114-136.
damage to cells.
Liposomes few nm
to few
µm [28]
1.Improve taste issues (e.g.
flavor encapsulation),
2.Provide good protection
to sensitive agents [28],
3. Can carry hydrophobic
and hydrophilic molecules
in the same cargo,
4.Mucoadhesive [28].
1.Most costly and complex technique
among the ones described in this review
2. It uses organic solvents [28],
3.Formulations are kept in dilution
aqueous solution,
4. Stability issues at high and room
temperature as well as low pH [28].
Coacervation 1µm to 1
Pectin [49],
gelatin [53]
1.High payloads (up to
99%) [46],
2.Mild preparation
3.High shell integrity.
1.Expensive technology,
2.Usual use of non-food grade
crosslinkers or enzymes, which are not
time efficient up to today [46],
3.Natural small differences in the
structure of the polysaccharides influence
the final capsule structure (quality control
Extrusion 100 µm
to 3 mm
Locus bean gum
[39], pectin [14],
chicory [54],
sugarbeet [54],
whey protein
[55], gelan gum
[56], xanthan gum
[56], gelatin [14],
chitosan [14], K-
carregeenan [51],
2.Scale-up potential,
3.Mild conditions,
4.Continous process
1.Frequently other technologies are
further used in order to obtain a final dry
2.Larger capsules.
2.1. Spray drying
This atomization technology is the most widely used encapsulation technique in the food
industry due to its continuous production feasibility and tailor-made characteristics depending on
feed and encapsulating agent [28]. The configuration of a basic spray dryer consists of a drying
chamber, which receives a liquid spray, and it is rapidly evaporated as soon as it encounters the hot
air flow (Figure 2). The liquid is broken into fine dried droplets. The air flow may be concurrent,
such as in the most food industry spray-driers allowing a rapid drying [59], but also mixed or
countercurrent [60]. Industrial equipment has evaporating rates in the range of 0.1 to 12 t/h.
Spray driers are used frequently to entrap oils, aroma, enzymes and pharmaceutical drugs. A large
amount of research has been put into encapsulating probiotic bacteria in a laboratory scale; however,
final cell viability in food carriers can still be an issue for industrial application due to temperature,
osmotic extremes and water activity (aw).
AIMS Materials Science Volume 3, Issue 1, 114-136.
Figure 2. Basic schema of a concurrent spray dryer.
Academia: Considerable amount of spray-dried probiotic papers are published every year.
Herein relatively recent work has been summarized. Yonekura et al. used sodium alginate, chitosan
and hydroxypropyl methylcellulose (HPMC) as co-encapsulants in the spray drying of L. acidophilus.
The authors report that chitosan reduces viable counts after spray drying whereas HPMC does not.
However, the former enhances storage stability and higher viable counts were achieved at a 35-day
storage period [43]. The study points out the importance of evaluating the viability during storage
and not only after the encapsulation procedure, since for industrial purposes, the cells protection is
essential during the storage.
Castro-Cislaghi et al. encapsulated B. animalis ssp lactis in milk whey. The viability of the
capsules remained high and constant (> 9 log CFU/g) for 12 weeks when stored at 4 °C [41].
However, the cells were not protected against the bile salts, which is fundamental for the final
application. Nevertheless, as also reported by Rodriguez-Huezo, the observed ‘flat ball effect
phenomenon provides the capsules with a certain mechanical robustness to mechanical fractures [61].
Behboudi-Jobbehdar et al. stabilized L. acidophilus in a maltodextrin/whey protein concentrate/
D-glucose carrier. The increase in the survival rate of the cells from 2.5 to 84% when the outlet
temperature was reduced from 91.5 to 60 °C was a meaningful finding [40]. In addition, the authors
concluded that a more elevated flow rate typically reduces cell membrane damage.
Ying et al. encapsulated L. rhamnosus in whey protein and/or resistant starch matrices followed
by a probiotic survival investigation over a 5-week storage period at 4 or 25 °C in citrate buffer (pH
of 3.5) or in apple juice. The reduction in cell numbers was greater for corresponding formulations in
citrate buffer than in the juice, where the pH also slightly decrease over time and cell density
increased [37]. L. rhamnosus is known to be an acid-resistant strain, and this work nicely indicates
that the strain can even better withstand acidic conditions when metabolizing substrates are available
in the media.
Corcoran et al. spray-dried L. rhamnosus in lag, early log and stationary phases and compared
the strain viability independently of the type of feed carrier used. The order of survivability is
AIMS Materials Science Volume 3, Issue 1, 114-136.
stationary, early log and lag with 50%, 14% and 2% respectively [62]. A unquestionably important
parameter that has to be considered at the beginning of the studies.
Finally, in order to aid the cells resist the dehydration process, osmoprotectants such as
trehalose [63], non-fat milk solids or adonitol [64] are added in the encapsulation systems. These
compounds influence the changes in the osmolality of the surroundings, the cell cytoplasm, and the
tension in the cell envelope [64].
Patents and Industry: The development of spray-dried probiotic bacteria has been limited in
large commercial scale to the best of our knowledge. Despite the high production rates and relatively
low operating costs, i.e. 4 to 7 times cheaper than freeze drying [38], most of the probiotic strains do
not survive in large quantities after the heat stress and dehydration. The high temperatures result in
high mortality and inactivation of microorganisms damaging cytoplasmatic membrane, cell wall,
ribosomes and DNA [59,65]. Another industrial limitation is the shell material, which needs to be
soluble in water at an acceptable level due to usual water aqueous formulations [46].
Table 2. Overview of key considerations when considering spray-drying technology for
probiotic encapsulation. Source: Information adapted from [38].
Type of bacteria
and stress tolerance
It is strain-dependent. Stress tolerance is an intrinsic physiological function.
Carrier Carriers are typically proteins
and/or carbohydrates.
They help bacteria reach their site of action with certain
Drying temperature The sensitiveness is strain-
Mortality increases with drying temperature and it is
accentuated typically above 51°C [67].
Survival rate was observed to be inversely proportional
Time of exposure to
Mortality is proportional to the
time of exposure.
Heating time should be as short as possible.
Osmotic, oxidative,
mechanical stresses
It is strain-dependent. Additives such as antioxidants or osmoprotectants can
be included. Pre-stressed bacteria before drying can
Glass transition Glassy state for food powder. Food powder should be in the glassy state to minimize
molecular mobility and avoid further detrimental
Barriers against the harsh
Coating enhances stability, especially during the
Water activity (aw),
moisture content
In order to ensure stability, aw
should be below 0.25. Moisture
content should be below 5% [66]
aw values between 0.11 and 0.23 prevented cell death
during storage and higher values were related to the
Storage conditions Microcapsules must be protected
from heat, oxygen and moisture.
Proper packing material must be
selected accordingly.
Survival is inversely related to storage temperature.
Decreasing order of spray-dried bifidobacteria survival:
vaccum laminated pouch > glass bottle > PET bottle
[69]. Vaccum storage was proven to be better than
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Stable spray dried culture starters are attractive to the fermented probiotic business as they
could be added directly into finished food, suppressing the need of liquid starter stocks and resulting
in less storage costs [66]. Table 2 exemplifies some aspects that should be considered to potentially
improve the chances of a successful spray drying encapsulation [38].
2.2. Spray cooling (or spray chilling)
The active ingredient is dispersed in a molten material, usually fats, serving as the carrier. The
cold air injected into the chamber enables the solidification of the microcapsules [71]. Customarily
spray cooling refers to a matrix encapsulation (Figure 1) due to the aggregation of the active
ingredients, which can also be found on the surface of the microsphere and therefore are readily
released [46].
The advantages of this technique are: (i) the least expensive with scale-up potential for
probiotics [44,46]. (ii) application of lower temperatures enabling its use with thermosensitive
microorganisms, (iii) capsules are generally insoluble in water due to the use of wax and oil as
carriers. Their solubility will depend on the hydrophilic and lipophilic characteristics of the
carrier [35]. On the other hand, few eventual hurdles are: (i) displacement of the core material during
storage due to the solidification and crystallization process of lipids [44], (ii) hydrophobic
characteristic of the material can be an issue for certain applications, (iii) difficulty to obtained a
delayed release of a water soluble ingredient over 30 minutes when the food carrier has high water
activity [46]. Enzymes, flavors, minerals and proteins have been encapsulated using this
technology [71].
Academia: This technique is less addressed in the probiotic field as far as our knowledge
concerning scientific papers available. Pedroso et al. microencapsulated B. animalis ssp lactis and L.
acidophilus with cocoa butter. The emulsion was formed using a high sheer device rotating at 9500
rpm for 60 s with subsequent atomization in a 10 °C chamber. Surprisingly the process conditions
did not impact negatively the cell viability. B. animalis ssp lactis free and encapsulated showed the
same resistance to simulated gastrointestinal fluids, however free cells were unstable during storage,
and 72% of the encapsulated cells remained viable after 90 days of storage at 7 °C. In the case of L.
acidophilus encapsulation increased its viability in simulated gastrointestinal fluid, but only 20% of
the cells remain viable in the above storage conditions [44]. When considering an industrial
application, this study confirms the relevance in choosing adapted strains in respect to the finished
product conditions.
Okuro et al. produced solid lipid microparticles with inulin or polydextrose containing L.
acidophilus. The polydextrose formulation presented the best cell viability with 6.9 or 6.8 log CFU/g
at relative humidity of 11% after 120 days of storage at 18 °C or 7 °C, respectively [45]. This
symbiotic system is an interesting deliver vehicle containing the minimum concentration of viable
cells required by the US FDA.
Patents and Industry: A shell-core type of microcapsule has slower release kinetics due to its
outer layer, often hydrophobic, which can be composed of single (Figure 1) or multiple shells. The
advantage of having shells lies on the ability to tune the wall thickness and customize the
microcapsule to the desired purpose. Such approach should be taken with careful judgment
considering the possible substantial inert material inclusion into the product matrix. Detrimental
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organoleptic effects and the impact in the nutritional-value of the product are eventual industrial
Rutherford et al. successfully developed a process of spray cooling using a rotary disc for the
animal feed field. The microspheres of fatty acid matrix (e.g. stearic acid) contain a plurality of
individual microorganisms, such as Enterococcus faecium, Lactobacilli or yeast. The microspheres
can be stored for a period of 3 to 6 months and cell viability is maintained even if there is exposure
to some moisture or antibiotics [72]. DuPont Pioneer owns the patent.
The expanding scenario of probiotics gains overtime more attention in the animal feed industry
as well where the rules for antibiotics administration become stricter and the intention of enhancing
animal health and productivity is a reality [73]. Lallemand Health Solutions commercializes
Bactocell®, a product based on viable Pediococcus acidilactici an additive that reduces bone
deformation in fishes [74]. Companies such as Performance Probiotics are entire dedicated to the
cattle feed business.
2.3. Freeze drying (or lyophilisation)
In a typical freeze-drying process, the liquid material containing the bacteria is first frozen with
further decrease of the chamber pressure enabling the frozen water to sublimate. It is an energetic
costly procedure, however there are at least two main advantages: (i) oxidation is avoided and, (ii)
the drying step is less damaging to the cells when compared to the high drying temperatures. An
eventual concern associated with this technique is the formation of ice crystals during the slow
freezing, which may lead into significant cell damage if not handled correctly [63]. In order to
attenuate cell loss, anti-freeze agents, known as cryoprotectants, are added to lower the freezing
point of water and consequently its vapor pressure [28]. Ideal cryoprotectants should be food grade,
permeate through the cell wall and contain non-toxic solutes. The synergistic effect of trehalose with
sugar alcohols, glycerol or certain proteins allows a superior stabilization of the bacteria mixture
during storage at long periods of time [75].
Academia: The technique of freeze drying is often utilized to preserve sensitive biological
substances and to date is the best process to dry bacteria due to the maintenance of the cells viability
during storage [3]. Nguyen at al. exposed B. bifidum, harvested in stationary phase, to 42°C for 100 s
and 300 s, and verified the strain increased its freeze drying resistance. The authors observed
changes in the zeta potential due to higher concentration of exopolysaccharides (EPS) secreted by the
bacteria after the heat shock [76]. EPS are known to induce stress resistance and in a way, stressing
the cells prior to freeze drying, increase their membranes robustness and as a consequence their
Basholli-Salihu et al. studied the effect of cellobiose, lactose, trehalose and sucrose, as
cryoprotectants in the survivability of B. infantis in milk (pH 6.7) and grape juice (pH 3.4) at 4
after freeze drying. All four protectants responded in a similar way during a 2-week storage in milk
with about no changes in viable counts in respect to time zero. However, cellobiose (5% and 10%)
and trehalose (10%) lost only one log after a 4-weeks storage in grape juice compared to time
zero [77]. If the storage of cells in low pH is of interest, then trehalose and cellobiose seem to be
better choices of cryoprotectants. Conrad et al. verified 14 years earlier the potential use of a
trehalose-borate system as cryoprotectant.[63]. The study suggests that borate can enhance this
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protective ability of trehalose. Nevertheless, borax, the borate source of the above study, is an illegal
food additive in several countries.
Hugo et al. confirmed the formation of stable cold-set gels with viable L. delbrueckii ssp lactis
when adding CaCl2 to high pressure-treated and freeze-dried soybean protein isolates. This
formulation could be interesting for the non-dairy probiotic market.
Ananta et al. reported sucrose as being the most effective cryoprotectant over betaine for B.
animalis ssp lactis and L. rhamnosus. Starch was also investigated as a carrier for its exceptional
touch in the final product. A dried product containing starch is granular and light in color allowing its
use in different food matrices. Nevertheless, its addition did not improve storage stability [66].
Patents and Industry: Oftentimes the high cost of this technique is highlighted as a drawback.
However, when considering the bacterial loss in the cost analysis of different drying methods, freeze
drying seems a competitive approach [28]. At present day the industry uses this technique to dry
components for value-added products, e.g. enzymes, probiotic pills for the reestablishment of the gut
flora and bacterial strains used in research. Traditionally cheesemakers purchase their freeze-dried
starter cultures from specialized suppliers [70].
2.4. Spray coating
Spray coating consists of forming a uniform layer onto solid particles. It is utilized with a vast
variety of shell materials such as polysaccharides, proteins, fats, yeast cell extract, or even complex
formulations. This versatility translates into potential controlled release features [46]. Despite the
three different air inlet positions, the basic functioning is the same. Powdered probiotic bacteria are
kept in motion in the chamber and a coating is sprayed over them. The final material will have
different characteristics depending on the applied spray coating configuration [78]. Fluidized bed
drying is a type of spray coating invented and patented in 1963 by Würster.
Academia: Several nutraceuticals, such as supplements and pills, are encapsulated using spray
coating. Schell et al. developed a microencapsulation system in a two-step process for L. reuteri
using sweet whey protein and dietary shellac. The bacteria slurry was first top-spray with sweet
whey along with trehalose, maltodextrin or sorbitol and a posterior shellac coating. Trehalose and
sorbitol gave the highest viability of encapsulated bacteria. In this study, shellac coated bacteria
survived best at pH 1.5 to 4.5 compared to only whey or free cells [79]. The uncommon use of
shellac in the probiotic encapsulation turns the study an innovative approach. This resin is secreted
by Laccifer Lacca, a female insect and it is FDA-approved to coat candies and fruits.
Semyonov et al. produced microcapsules containing L. paracasei using a Würster coater system
with a bottom-spraying atomizer. The authors investigated the use of different coatings.
Hydrogenated vegetable oil provided excellent moisture protection with 99% of cell survivability
and ethylcellulose conferred protection during the gastrointestinal passage. The inlet air temperature
was the most detrimental parameter with a 250-fold decrease in survival rate when a 15 °C
temperature increment was applied [80].
Bensch et al. investigated the effects of fluidized bed drying on the viability of L. plantarum ssp.
plantarum via flow cytometric and plate count methods. The difference shown between both
methodologies is suggested by the authors as an occurrence of viable but nonculturable cells during
storage [81]. Flow cytometry has potential for a rapid routine in-process industrial control while
plate count is a laborious and time-consuming technique.
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Champagne et al. evaluated various homogenization, rehydration and plating practices of
freeze-dried L. rhamnosus and B. longum, which were spray coated with fractionated palm oil and
palm kernel oil. This study points out that L. rhamnosus has a better survival rate to freeze drying
than B. logum and spray-coated cultures rehydrated slower and only partially [82]. The rehydration
may be a crucial factor when the encapsulated material is blended with a given product.
Patents and Industry: Fluidized bed drying is commonly used in the food industry. This
technology allows the use of milder temperatures compared to spray drying due to optimal heat and
mass transport resulting in higher cell survival [79].
Wu et al. coated lactic acid bacteria with high laurate canola oil (HLCO) holding a low melting
point. Not only higher yields of encapsulated bacteria were observed when compared to the use of
hydrogenated vegetable oils, but the use of HLCO lower temperature reduces smearing, caking and
doughing up [83]. As a consequence, a product containing no cracks with a free flowing profile is
homogeneously produced.
Ubbink et al. invented a probiotic delivery system where 0.02 cm3compacted pellets containing
probiotics and further fillers improved the stability of the microorganisms when applied in semi-dry
and/or humid particulate foodstuffs [84]. Enterococcus faecium has about 80% of viable cells
recovery after a 30 days storage period at 30°C in 70% relative humidity. The patent belongs to
Nestlé SA.
Lallemand Health Solutions has developed an encapsulation technology named Probiocap®.
The molten hydrophobic coating, a mixture of at least one fatty acid and/or wax, is injected in a
rotational vessel containing agglomerates of living dehydrated probiotics [85]. One of the advantages
of the process includes suitability with a wide range of coating materials, which can have a melting
point greater than the temperature of the human body avoiding an early stage disintegration of the
capsules. The same company developed Star®, a water-based enteric-coating with excellent barrier
protection that allows manufactures to add a lower concentration of microorganisms per capsule.
2.5. Emulsification
An emulsion is a mixture of two or more immiscible liquids where one liquid (the dispersed
phase) is dispersed in the other (the continuous phase) under the stabilizing effect of surfactants. The
most common emulsions are water-in-oil (w/o), oil-in-water (o/w) and water-in-oil-in-water (w/o/w).
Emulsion-based processes have a drawback in regard to the difficulty in controlling the size
distribution of the droplets. In order to produce monodisperse droplets, there are two emerging
technologies: membrane emulsification and microfluidic systems. The use of low sheer stresses with
these techniques makes them suitable for sensitive compounds.
Academia: Emulsion-based techniques are on the radar of many researchers. Mantzouridou et al.
studied inulin-based emulsions. L. paracasei ssp paracasei was either suspended in the aqueous
phase or entrapped in corn oil droplets of three emulsion systems containing egg yolk, gum
arabic/xanthan or whey protein isolate. The free cell control in the aqueous phase and stabilized with
egg yolk had the highest cell viability after a 4-week refrigerated storage when compared to the cells
imprisoned in oil droplets. Inulin enhances the stability of the emulsion by providing stronger
interactions with the oil droplets, and it seems to support probiotic culture growth [50].
Amine et al. studied the survival of B. longum in cheddar cheese in terms of freezing tolerance,
storage in a frozen state, cheese manufacturing and stimulated gastrointestinal conditions. The
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technologies of co-axial airflow extrusion and emulsion along with the use of native and
palmitoylated alginate as encapsulating agents were exploited. A 30-days storage at 80 °C showed
similar viable counts for free cells and both encapsulation systems when utilizing either alginate [52].
The alginate confers protection mostly during gastrointestinal passage and storage of the cheese.
Dianawati et al. prepared fifteen different emulsion systems to evaluate the survival of B.
longum after freezing steps. Sodium caseinate, whey protein concentrate, sodium caseinate:whey
protein concentrate, skim milk, soy protein were combined with glycerol, mannitol or maltodextrin.
Overall milk proteins and sugar alcohols were more effective than soy protein isolate and
maltodextrin [48]. One explanation may be due to the easier interaction of the hydroxyl groups of
sugar alcohols with the polar sites of phospholipid bilayers composing the cell membranes.
Patents and Industry: Herein some interesting patents based on emulsion techniques. Beck et al.
elaborated a w/o/w edible emulsion containing probiotic bacteria in the internal water phase. The
emulsion has an acceptable level of viable cells after 3 weeks of storage at 8 °C [86]. A particular
advantage is that it holds no volume restrictions in respect to the concentration of probiotics that can
be added to the internal phase. The invention relates to an emulsion for salad dressings and belongs
to Unilever.
Vos et al. created a method to produce stable w/o/w emulsion useful to probiotics. It holds the
advantage of masking the flavors of the aqueous components of the inner water phase and
maintaining their stability for longer [87]. The emulsion components are common in the academia
studies such as sunflower oil and skimmed milk powder except for admul (an emulsifier) and
hipotral 35 (a whey protein source).
Mazer et al. patented a method for producing a stable emulsion that is further added into a
powdered nutritional product designed to infants, children or adults. Any acidophilic and/or
bifidobacteria can be incorporated into the method [88]. Abbott GmbH holds the rights on it.
The size distribution of emulsion droplets produced by simple high sheer methods is often seen
as a drawback. Nowadays it can be solved by the microfluidic technology. There are at least two
companies that use the microfluidic systems to manufacture monodisperse droplets. Nanomi
manufactures injectable pharmaceutical products and Micropore® Technologies transforms their
knowledge into application in different fields, such as nutraceuticals delivery or chromatography.
The microfluidic technique has tremendous size distribution homogeneity; however, the high cost of
final product is still a hurdle for the production of ordinary foodstuff.
2.6. Liposomes
Liposomes are spherical bilayers composed of phospholipids similar to cellular membranes.
Hydrophilic molecules are entrapped in the water-soluble interior of the liposome, and hydrophobic
in the oil-like portion. A potential use of liposomes lies on using one single cargo to delivery
hydrophilic and hydrophobic active ingredients. The large unilamellar vesicles (LUV) are the most
pertinent for the food industry due to their high encapsulation efficiency, straightforward production
methods and over time better stability [46]. Main drawbacks of such technique are upscaling costs
and often the formulations are kept in rather dilute aqueous suspensions, which translate into
additional costs [46].
Academia: Liposomes are used in a diversity of products in the beverage, cheese industries and
in the elaboration of vaccines. Enzymes, small molecules and antimicrobials are some of the
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examples in which this technique is used [28]. To the best of our knowledge, little has been done
currently in the probiotic field. Nevertheless, giant liposomes having a diameter greater than 1 μm
are of particular interest in the encapsulation of bacterial cells, which possess a size ranging between
1 and 4 µm. Their stabilization may be an issue along with over time oxidation, aggregation and
Patents and Industry: There are still several issues to be addressed in order for liposomes to be
successfully utilized in the probiotic industry. Chemical degradation, stability and oxidation are main
downsides. The use of liposomes in value-added products is however current and well established in
the pharmaceutical industry. For the beverage industry, one smart development to package liposomes
and avoid the lipid degradation is by freeze drying it. Curcusome®, a freeze-dried liposomal
formulation of curcumin/quercetin/piperine can be kept in release caps. Since liposomes are not
stable at low pH or high temperatures, often parameters of the beverage industry, the liposomal
powder is released from the cap into the beverage right at consumption. Innovations as such can
easier bridge the liposomes research to probiotics.
Frenken et al. published a patent for treating, reducing or preventing diarrhea and treating
rotavirus infection. The food or pharmaceutical preparation has antibodies, antibody fragments and
probiotics. The eventual invention may be an additive in margarine, ice-cream, dressing or even
beverages [89]. Unilever owns the rights.
Gregoriadis et al. patented a method for the formation of liposomes with a vesicle diameter
smaller than 50 μm containing one or more entrapped Bacillus subtilis. After the formation of it, the
liposome is freeze dried. The cell viability maintains stable after the dehydration/rehydration
processes [90].
2.7. Coacervation
It is a well-developed and widely studied technique applied with several hydrocolloid systems
such as gelatin/acacia gum, carrageenan, chitosan, soy protein and gelatin/carboxymethylcellulose.
Coacervation can achieve rather high payloads, on the other hand the process cost is expensive [28]
and frequently crosslinking substances are involved, such as glutaraldehyde or enzymes [46]. One
(simple coacervation) or more (complex coacervation) hydrocolloids are dispersed in an aqueous
solution with an active substance. With a pH change, the opposite charged colloids link together
forming a layer around the active substance.
Academia: This technique is more of a matrix type (Figure 1), which means the bacteria might
be present on the surface of the microsphere. Gerez et al. encapsulated L. rhamnosus combining
ionotropic gelation using pectin and pectin-whey protein and further complex coacervation of whey-
protein forming a coating without involving the use of organic solvents. Electrostatic complexes are
formed below the isoelectric point of the whey protein (pH 5.4) where mainly positively charged
NH3+ of the whey protein is attracted by the carboxylate groups of the pectin. The authors observed
an effective protection of L. rhamnosus with the pectin-whey protein formulation resulting in a
survival rate of 107 CFU/ml in pH 2 [91].
Shoji et al. microencapsulated L. acidophilus by complex coacervation employing pectin and
casein followed by spray drying and further addition it to buffalo milk yogurt. The bacteria survived
in 107 CFU/g in buffalo milk presenting a shelf life of around 120 days at 7 °C. However, adequate
amounts did not survive the simulated pH conditions of the stomach [49].
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Patents and Industry: The complex coacervation has been used widely to encapsulate flavors
and unsaturated fatty acids in the industry with the goal of enhancing their shelf life. Bazo Agüeros
et al. developed a method for the encapsulating of probiotic bacteria producing microparticles with a
matrix composed of casein and chitosan. The capsules size is controllable with diameters below 100
μm [92]. This patent is an example of eventual industrial feasibility of the coacervation method for
the encapsulation of probiotics. The inventors were able to obtain capsules with sizes in agreement
with the food industry requirements.
2.8. Extrusion
There is a considerable amount of extrusion methods, such as dripping by gravity, electrostatic
potential, vibration for jet break-up, JetCutter, and others [59]. This technology holds the basic
concept of dispersing the bacteria in a solution that goes through a dripping nozzle and the droplets
harden simultaneously by gelation when contacting with a second solution.
Electrospraying is a microencapsulation emerging technology and relies on the application of
high potential electrical field to obtain spheres. The polymer solution is extruded from a capillary
where two major electrostatic forces are acting: the repulsion of like charges and the Coulombic
forces of the external electrical field. At the tip of the needle, the droplet is distorted in a conical
shape, and once the electrostatic forces counteract the surface tension of the bead, a charged droplet
is ejected from the tip of the cone [93]. Vibration for jet break-up and JetCutter will break the stream
of liquid with vibration and a rotating cutting tool, respectively. The formation of uniform beads and
the use of mild temperatures are an advantage of the extrusion techniques.
Academia: Extrusion is largely applied in research and sometimes considered as a laborious
technique. However, with the development of different technologies, which go beyond the batch
processes, industrial applications should be on the rise. Borges et al. evaluated the effect of
microencapsulation on the viability of L.casei, L. paracasei, L. acidophilus and B. animalis in an
alginate matrix. The effects of L-cysteine addition, exposure to pH of 3, NaCl (25% w/v) and
temperatures of 55 °C and 60 °C were investigated. Results concluded that survival is strain-
dependent. Free and encapsulated L. acidophilus demonstrated the highest survival rates when lethal
conditions of temperature and pH were applied [94]. The authors discuss the final bead size and its
protection role. The requirement of the food industry to avoid organoleptic drawbacks in a given
product is of 100 µm and in fact humans can detect particles of sizes small as 25 µm [95]. However,
smaller sizes confer less protection. Finally, the balance between final size and intended protection is
debatable and it will ultimately depend on the characteristics of the food carrier.
Nualkaekul et al. compared alginate and pectin beads for improving the survival of L.
plantarum and B. longum during storage in pomegranate and cranberry juices. The influence of
various coating materials including chitosan, gelatin and glucomannan on cell survival was assessed.
The double gelatin coated pectin beads coffered the highest protection in the juices. The effect may
be attributed to the formation of a dense polyelectrolyte complex, which eventually increased the
buffering effect of the beads. For instance, the coating protected the beads against the penetration of
gallic acid [14]. It is a very important result since there are large numbers of studies using the
alginate/chitosan system, yet chitosan is allowed in the pharmaceutical but not in the food industry at
present time as far as our knowledge.
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Khan et al. entrapped B. adolescentis in capsules composed of biopolymer mixtures of chickpea,
faba, lentil or pea protein isolates with alginate. Shelf life studies with plain yogurt as a model
showed a reduction of 3 log10 CFU/ml for entrapped cells with all capsules designs in a period of 7
days whereas free cells had 8 log10 CFU/ml reduction [58]. The use of legume proteins along with
alginate is novel and this approach opens up the ingredient market to non-dairy applications and,
consequently, to a different customer niche.
Laelorspoen et al. investigated the use of electrospraying in a zein-alginate core-shell system.
When increasing the voltage from 4 to 10 kV, the capsules size decreased from 543 to 259 µm and
the viable counts of L. acidophilus suffer only a minor change from 8.85 to 8.31 log CFU/ml,
respectively [96]. The electrospraying technique for bacteria encapsulation shows therefore a great
potential. The mild conditions used along with scale-up possibilities are indeed positive aspects that
should certainly attract more attention for this technique in the near future.
De Prisco et al. used a vibrating technique to encapsulate L. reuteri in alginate/chitosan matrixes.
The authors report a bead size of 110 ± 5 µm and encapsulation efficiency higher than 90 % for the
four systems studied. They also report a 100% of cell survival after freeze drying with no changes on
the structure of the beads. The very narrow size distribution reported is an important parameter for
homogeneous release of the probiotic in the target site. Additionally, the high survivability of the
cells with no changes in the bead structure after freeze drying confirm the efficacy of this drying
technique to keep the final material in a powder form [97].
Graff et al. encapsulated Saccharomyces boulardii in an alginate/acrylic acid matrix coated with
chitosan using the laminar jet break-up. The use of acrylic acid minimized the aggregation and
sticking of the beads. In this study the narrow size distribution of the beads was also verified as in the
studies of De Prisco et al. confirming the potential of this technique for industrial purposes [98].
Patents and Industry: Extrusion of active ingredients represents only up to 3% when compared
to other technologies in the food industry [28].
BRACE GmbH proposes a vibrating nozzle system for the encapsulation of a variety of
ingredients. The company holds a patent for production of spherical alginate beads in industrial
scale [99]. Based on the BRACE technology, Vésale Pharma developed Intelicaps®, uniform
probiotic microcapsules with an alginate-based shell and typical sizes of 600 to 800 µm. The layer-
by-layer coating reinforces the pH responsiveness of the system resulting in bead disintegration at
pH 5.5 to 6.
Van invented a continuous process for producing controlled release particles containing
sensitive components. The particulates are produced at low temperatures and the encapsulation
process increases the stability of the active ingredient against moist heat [100], which may be a
parameter in the downstream of the industrial processing. Patent owned by General Mills.
Morishita Jintan Co. Ltd produces Bifidobacteria capsules of 2 mm with a three-layer shell of
gelatin to be blended with yogurt. The company owns the patent of Asada et al. who created a
multilayer capsule composed of cells capable of growing in the confined state. The authors suggest
its use in food for treating intestinal disorders or as bioreactors [101].
Doherty et al. produced round droplets of gelled whey protein via the vibrational nozzle
technique with further immersion of the beads in an acidic curing solution [102]. The innovative
academic work became a start-up, AnaBio Technologies, a company that provides encapsulation
expertise for the food, pharmaceutical and animal health care products.
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3. Final Valuable Remarks
Encapsulation Interest: Frequently probiotic products, often yogurt, available in the market
retain little cell viability at time of consumption. Such viability is strain-dependent and depends on
interactions with other species present in the product, culture conditions and production of
metabolites affecting the final acidity of the product. In the past, several reports have drawn the
attention of scientists to the poor survival of strains in ready-to-consume yogurts [103]. Three out of
six samples in a dairy product study contained no traces of live bacteria [28]. These insights
highlight the importance of providing protection to bacteria strains before addition to food carriers
and consumer consumption.
Cultivation Medium: The most widely used growth medium in academia research to cultivate
Lactobacilli is the MRS. Despite its effectiveness, the high cost of the ingredients and the non-food-
grade label of some of its components turns its use in the industry less of a likelihood. A universal
medium called GEM containing just food grade ingredients and composed by glucose, soy peptone,
yeast extract, MgSO4 x 7 H2O and KH2PO4 was elaborated by Saarela [66]. The colony forming
units (CFU) obtained with GEM were the same or even better than with MRS. For Lactobacillus
rhamnosus, a supplement of Tween 80 was added, which resulted in better stability during freeze
drying and further storage at 37 °C [66].
Industrial secrets and patents: Although many patents are in circulation and can be potentially
used by academic researchers to get an industry trend sense, it is of importance to note that there are
many unclosed facts within their judicious written language. As a fact, knowledge is transmitted with
a patent, but its occurrence deals mostly with legal rights.
Production scale and costs: The production scale and commercialization value of encapsulated
ingredients needs to be evaluated when intention of extending academia results into industrialized
products is a reality. Continuous or batch production is one of the very first key cost-effective
parameters to be taken into consideration. For instance, spray drying can continuously produce
microcapsules; on the other hand, emulsion-based techniques are conventionally batch processes.
The prices of the technology and raw materials should be also put into question; and a great
challenge arises from it. As an example, the customary studied alginate and proteins systems have a
low solubility in water and require a costly energetic input to be assessed in the industry [42,46,104].
Product development is also expensive and time-consuming, and generally low margins are achieved
with food ingredients in the food industry [46]. Additionally, encapsulation adds another step in the
food-processing stream [104]. These are major reasons for higher prices of encapsulated probiotic
products. In the end, encapsulated probiotic products are developed for a specific niche market: those
who are willing to pay for it.
4. Conclusion
A multitude of research has been done on trying to find suitable polymer matrices along with
process technologies. Commonly, natural occurring proteins and polysaccharides are a good polymer
choice due to their non-toxicity and biodegradability. The microcapsules must be finely tuned to
achieve important goals for the food delivery systems, such as heating and freezing robustness,
storage stability and bile and acid-tolerance. Long-term storage imposes a main obstacle for
encapsulation of bacteria due to cells subproducts release overtime or lack of nutrients to keep them
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alive. Challenges in the fabrication of microcapsules are still manifold and ultimately related to the
complexities of bacteria and their response under confinement.
The spray drying, emulsification and extrusion techniques are heavily utilized in the probiotics
academic research. The spray drying technique allows the production of small capsules that can be
mass-produced, however it damages the cells due to the high temperature applied during the process
resulting in poor final cell viability. Emulsification can be scaled up but it can be a costly process
that produces capsules varying in size and shape, another drawback. Extrusion, however, utilizes
oftentimes mild conditions and the production of narrow size capsules is possible. Electrospraying is
gaining more attention for instance, and the vibration nozzle technique is already in the market.
Spray coating and spray chilling are continuous processes and overall cost-effective. Both
technologies work with mild temperatures translating into fewer bacterial cell injuries. As far as
drying techniques, freeze drying under vacuum with cryoprotectants has an interesting potential due
to favorable low temperature exposures and superior contamination control. The elevated price of the
technique diminishes when cost analysis considers the cell loss in other drying techniques.
Generally speaking, the cost of using encapsulated bacteria in the food industry is still a hurdle
and in addition, still a low payload of viable bacteria reaches the gastrointestinal tract. Advances in
encapsulation should bridge this gap in the near future. Along these lines, the collaboration between
academia and industry could result into successful market novelties.
The research of the gut microbiota is on the radar of different domains. Recent discoveries
indicate the impact of the intestine bacteria in respect to the brain functioning and its influence in
disorders such as anxiety and depression [105]. Scientists are only starting to understand the
diversity, dynamic nature and distribution in this microbiome. Ongoing and future research will
contribute to further elucidate the relationship between gut-microbiota and mental disorders. As a
result, meaningful novel approaches for their prevention and treatment should emerge one day.
The authors of this paper have received funding from the People Program (Marie Curie Actions)
of the European Union's Seventh Framework Program FP7/2007-2013/ under REA grant agreement
n° 606713 BIBAFOODS.
Conflict of Interest
All the authors declare no conflict of interest in this paper.
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... However, low encapsulation capacities on the beads produced through this technique have been reported. A lower load (10-20%) was obtained when compared to spray-drying (5-50%) [64]. The beads produced through the spray chilling technique have low melting points (32-42 °C). ...
... Special storage conditions can be required. [43,44,48,64] Fluidized bed coating ...
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The growing health awareness among consumers has increased the demand for non-dairy-based products containing probiotics. However, the incorporation of probiotics in non-dairy matrices is challenging, and probiotics tend to have a low survival rate in these matrices and subsequently perform poorly in the gastrointestinal system. Encapsulation of probiotics with a physical barrier could preserve the survivability of probiotics and subsequently improve delivery efficiency to the host. This article aimed to review the effectiveness of encapsulation techniques (coacervation, extrusion, emulsion, spray-drying, freeze-drying, fluidized bed coating, spray chilling, layer-by-layer, and co-encapsulation) and biomaterials (carbohydrate-, fat-, and protein-based) on the viability of probiotics under the harsh conditions of food processing, storage, and along the gastrointestinal passage. Recent studies on probiotic encapsulations using non-dairy food matrices, such as fruits, fruit and vegetable juices, fermented rice beverages, tea, jelly-like desserts, bakery products, sauces, and gum products, were also included in this review. Overall, co-encapsulation of probiotics with prebiotics was found to be effective in preserving the viability of probiotics in non-dairy food matrices. Encapsulation techniques could add value and widen the application of probiotics in the non-dairy food market and future perspectives in this area.
... Additionally, probiotics have shown anti-mutagenic potential (Yu and Li 2016), anti-carcinogenic properties (Casas-Solís et al. 2020;Devaraj et al. 2019;Wollowski, Rechkemmer, and Pool-Zobel 2001) and anti-diarrheal potential (Devaraj et al. 2019;Liu et al. 2017), preventive role of atopic dermatitis (Huang et al. 2017;Lise, Mayer, and Silveira 2018;Rather et al. 2016), a significant reduction of blood cholesterol levels (Nath et al. 2018;Shimizu et al. 2015), an antibacterial competitive properties between beneficial and pathogenic bacteria (Piewngam et al. 2019), a biosorption of toxins (Zoghi, Khosravi-Darani, and Sohrabvandi 2014;Zoghi et al. 2017) and heavy metals Hadiani, Khosravi-Darani, et al. 2018) from foodstuff. To get the beneficial effects of probiotics, a daily intake of 10 7 -10 9 Colony Forming Units CFU/g of probiotic viable cells per person for humans is recommended (Haffner, Diab, and Pasc 2016). ...
The world of probiotics and their applications have witnessed an unprecedented growth in terms of benefactions in various fields in order to promote the gut health as well as to alleviate certain clinical ailments associated with microbial dysbiosis. However, the coin had shown, in its other side, several theoretical risks in case reports, clinical trial results and experimental models, encompassing health risks. The safety of probiotics is firmly tied to their intended use, which comprises consideration of potential susceptibility of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. In this chapter, we tempt to unravel the secret of the double-edged sword of these beneficial microbes with an exhaustive discussion of probiotics safety, their regulation in addition to many other features and attributes of these beneficial microorganisms. Hence, while the overwhelming existing evidence suggests that probiotics are safe, complete consideration of risk-benefit ratio before prescribing is crucially recommended.
... Additionally, probiotics have shown anti-mutagenic potential , anti-carcinogenic properties (Casas-Solís et al. 2020;Devaraj et al. 2019;Wollowski, Rechkemmer, and Pool-Zobel 2001) and anti-diarrheal potential (Devaraj et al. 2019;Liu et al. 2017), preventive role of atopic dermatitis (Huang et al. 2017;Lise, Mayer, and Silveira 2018;Rather et al. 2016), a significant reduction of blood cholesterol levels (Nath et al. 2018;Shimizu et al. 2015), an antibacterial competitive properties between beneficial and pathogenic bacteria (Piewngam et al. 2019), a biosorption of toxins (Zoghi, Khosravi-Darani, and Sohrabvandi 2014;Zoghi et al. 2017) and heavy metals Hadiani, Khosravi-Darani, et al. 2018) from foodstuff. To get the beneficial effects of probiotics, a daily intake of 10 7 -10 9 Colony Forming Units CFU/g of probiotic viable cells per person for humans is recommended (Haffner, Diab, and Pasc 2016). ...
... 6 An industrially scalable solution to produce robust bacterial inoculants in a dried product is encapsulation in a cross-linked alginate matrix via a novel spray drying process. 7−10 Although spray drying has proven to be an effective method of encapsulation for probiotics and Gram-positive bacteria in the pharmaceutical industry, 11,12 Gram-negative bacteria are notoriously difficult to preserve thanks to their lack of a thick peptidoglycan layer relative to their Gram-positive counter parts. This makes them particularly susceptible to drying processes, including high temperatures, 12,13 shear, and osmotic stresses. ...
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Microbial inoculants are environmentally sustainable alternatives to petrochemical-derived herbicides, pesticides, and fertilizers in agriculture. We demonstrated the potential to develop a commercializable Klebsiella variicola product with storage and on-seed stability by encapsulating in cross-linked alginate matrix (CLAM) powder by spray drying. The cross-linked alginate microencapsulation by spray drying ( CLAM process'), an industrially scalable one-step strategy, produced a K. variicola-loaded CLAM with up to ∼1 × 1010 CFU/g powder. The K. variicola-loaded CLAM maintained high viability during encapsulation, slurrying, and adhesion onto seed surfaces (∼7 × 105 CFU/seed) and during ambient storage on corn seeds. K. variicola in CLAM powder maintained long-term (>5 months) viability in controlled environments, such as refrigerated, or oxygen and moisture-free environments. Trehalose with maltodextrin when added to the CLAM successfully extended stability in powder under ambient conditions, while maltodextrin alone had limited impact. Encapsulation in the CLAM maintained K. variicola viability on bare corn seeds with storage stability beyond 3 months.
Food products contain various active ingredients, such as flavors, nutrients, unsaturated fatty acids, color, probiotics, etc., that require protection during food processing and storage to preserve their quality and shelf life. This review provides an overview of standard microencapsulation technologies, processes, materials, industrial examples, reasons for market success, a summary of recent applications, and the challenges in the food industry, categorized by active food ingredients: flavors, polyunsaturated fatty acids, probiotics, antioxidants, colors, vitamins, and others. We also provide a comprehensive analysis of the advantages and disadvantages of the most common microencapsulation technologies in the food industry such as spray drying, coacervation, extrusion, and spray cooling. This review ends with future perspectives on microencapsulation for food applications.
Probiotics are well known for supporting a healthy digestive tract and aiding nutrition digestion in the human body. Bioencapsulation is a great way to boost the protective impact, extend the shelf life, and deliver probiotics to the human gastrointestinal tract at a specified moment. As a natural delivery strategy for food applications, probiotics are frequently entrapped in a biopolymeric system or the food itself. Probiotics encapsulated with natural biopolymers such as alginate, chitosan, gum Arabic, gelatin, starch, and whey protein improve their release properties, enhance healthy microbiota in the gastrointestinal system, and reduce emerging pathogens. This chapter will highlight the types of wall material used for bioencapsulation, the various types of bioencapsulation techniques, mechanism of bioencapsulation, and characteristics and properties of encapsulated probiotics, including the stability, bioavailability, and release properties. At the end of this chapter, the challenges and safety issues related to the bioencapsulation of probiotics are discussed.
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Probiotics are known as the live microorganisms which upon adequate administration elicit a health beneficial response inside the host by decreasing the luminal pH, eliminating the pathogenic bacteria in the gut as well as producing short chain fatty acids (SCFA). With advancements in research; probiotics have been explored as potential ingredients in foods. However, their use and applications in food industry have been limited due to restrictions of maintaining the viability of probiotic cells and targeting the successful delivery to gut. Encapsulation techniques have significant influence on increasing the viability rates of probiotic cells with the successful delivery of cells to the target site. Moreover, encapsulating techniques also prevent the live cells from harsh physiological conditions of gut. This review discusses several encapsulating techniques as well as materials derived from natural sources and nutraceutical compounds. In addition to this, this paper also comprehensively discusses the factors affecting the probiotics viability and evaluation of successful release and survival of probiotics under simulated gastric, intestinal conditions as well as bile, acid tolerant conditions. Lastly applications and challenges of using encapsulated bacteria in food industry for the development of novel functional foods have also been discussed in detail too. Future studies must include investigating the use of encapsulated bacterial formulations in in-vivo models for effective health beneficial properties as well as exploring the mechanisms behind the successful release of these formulations in gut, hence helping us to understand the encapsulation of probiotic cells in a meticulous manner.
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Probiotic bacteria such as Lactobacillus rhamnosus GG (LGG) play an important role in maintaining a healthy microbiota environment. Oral administration is the only way to deliver probiotic bacteria to the small intestine. Due to the harsh environment of the gastrointestinal tract, only a small proportion of probiotic bacteria can reach the target site, limiting their beneficial effects. Although many encapsulation techniques exist, the loss of bacterial cell viability, high cost, and low payload remain a major problem. In this work, we present a state-of-the-art microextrusion technique for encapsulating probiotic bacteria using a 3D bio-printer. Using a composite of sodium alginate, gelatin gel and LGG as bioink, we printed encapsulated LGG bacteria that were stable for 7 days. The production of the encapsulated bacteria was a very fast and simple process. We printed 120 probiotic capsules, each containing 10⁸ CFU/ml, within 30 minutes. On microscopic examination of the LGG capsules, the encapsulated LGG bacteria were evenly distributed in the alginate-gelatin hydrogel. The bacteria in the capsules were viable and alive for 7 days at 4°C and survived the harsh gastrointestinal fluid environment in vitro. This bioprinting method for encapsulating probiotic bacteria for their specific delivery in the gut could become a revolutionary method for industrial applications.
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Probiotics frontier in depressing the clinical bacterial pathogens to avoid multidrug resistance phenomenon. The present study aimed to determine the antibacterial efficiency of chitosan encapsulated probiotics isolated from buffalo milk samples against clinical bacterial pathogens. The Agar well method was used for antibacterial activity. Lactococcus lactis (A) and Lactobacillus curvattus (B) were isolated from fresh buffalo milk samples, identified via culturing media, Gram’s staining, biochemical tests, and antibiogram analysis. Encapsulation of probiotics was carried out using chitosan and was characterized via a scanning electron microscope. Antibiogram analysis elicit that L. lactis culture (A1) was highly sensitive to chloramphenicol (17.66±0.47 mm), tobramycin (15.33±0.47 mm), and ciprofloxacin (12.33±0.47 mm) and resistant against tetracycline, Penicillin G, Erythromycin, Amoxycillin, Ceftriaxone, Cephalothin, and Cephradine, while L. curvattus culture (B1) was affected by Ceftriaxone (18.67±0.47 mm), Amoxycillin (14.33±0.94 mm), Cephalothin (13.67±0.47 mm), Erythromycin (13.33±0.47 mm), Penicillin G (12.67±0.47 mm), Cephradine (10.33±0.47 mm), and Chloramphenicol (9.67±0.47 mm) and resistant against tetracycline, Tobramycin, and Ciprofloxacin. Antibacterial efficacy of non-encapsulated probiotic cultures was significant and maximum inhibition of bacterial were recorded compared to their cellular components. SEM of encapsulated probiotics revealed that they were successfully covered with a chitosan protective layer and could be effective as bio-preservatives due to being slowly released at the target site. The current study concluded that L. lactis, L. curvattus, and their cellular components have a significant bactericidal effect against infectious pathogens and could be used as a potential therapeutic drug against infectious diseases. graphical abstract Fullsize Image
Background. The probiotics immobilization technology is one of the most effective ways for controlled and continuous delivery of viable cells into the intestine. It is well known that multifaceted physiological roles of bifidobacteria are to normalize and stabilize the microbiocenosis, to form intestine colonization resistance, to synthesis amino acids, proteins and vitamins, to maintain non-specific resistance of the organism and so all. Such a wide range of positive effects on the macroorganism allows us to consider bifidobacteria as a basis for functional immobilized healthcare products development. Objective. Taxonomic position determination of the Bifidobacterium longum strain selected for immobilization, study of the viability of this bifidobacteria strain in a complex probionic product based on highly dispersed silica in simulated gastrointestinal tract's conditions and after freeze-drying. Methods. The production strain Bifidobacterium longum IMV B-7165 from the Institute of Food Resources of the National Academy of Agrarian Sciences of Ukraine collection of industrial strains has been used in the study. It was isolated from the healthy human infant's gastrointestinal tract. Commonly used bioinformatics, microbiological, biotechnological and statistical methods have been applied. Results. The best alignments for the sequence of bifidobacteria isolate "4202" 16S rRNA (it was previously deposited as Bifidobacterium longum IMV B-7165) and classic dendrograms based on these results were performed. According to the results of microscopic studies of samples of microorganisms with highly dispersed silica products ("Enterosgel", "Sillard P" and "Toxin.Net") it was found that the immobilization of the Streptococcus thermophilus and bifidobacteria cultures did not differ fundamentally. To study the immobilization effect on the bifidobacteria preservation and properties the following carriers were used: "Enterosgel", "Toxin.NET" and "Sillard P". The survival of immobilized bifidobacteria was further studied in simulated gastrointestinal conditions: immobilized cells are better protected from acid and bile, although with increasing acidity, survival decreases in both control and immobilized cells. Conclusions. The taxonomic position of a bifidobacterial isolate from the healthy human infants used in immobilization studies was clarificated (Bifidobacterium animalis subsp lactis). Under the simulated conditions of the upper gastrointestinal tract in the case of acid and bile impact, the best survival was demonstrated by immobilized cultures of bifidobacteria together with the Enterosgel sorbent (a content of 10% by weight of the culture). The survival of immobilized preparations after freeze-drying was slightly reduced in the case of immobilization on the "Enterosgel" and "Toxin.NET" samples of enterosorbents (a content from 15% to 25% by weight of the culture). The best results were observed in the case of immobilization of bifidobacteria with 5% content of the "Toxin.NET" enterosorbent (enterosgel + inulin).
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Artificial seeds formed of a seamless soft capsule are provided herein. The artificial seed contains a meristematic plant cell or tissue. The artificial seed has a structure of three or more layers containing an innermost layer, an inner layer covering the innermost layer, and an outer layer covering the inner layer. The innermost layer can contain an aqueous liquid, the inner layer can be formed by an inner membrane containing hardened oil, and the outer layer can be formed of a biodegradable outer membrane.
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The feasibility of vacuum impregnation in combination with air drying + radiant energy vacuum (REV) drying to produce shelf stable probiotic enriched apple slices was evaluated. The shelf life of the products was monitored at 25 and 4 degrees C. The results demonstrated that bacterial stability at 25 degrees C depended on the dehydration techniques; with the longest shelf life in air drying +REV drying followed by freeze drying and air drying. Storage at 4 degrees C showed no significant changes in bacterial population up to 180 days in all samples. Sensory properties of the air drying +REV and freeze dried apple slices remained above the acceptable level for 30 days at 25 degrees C and 180 days at 4 degrees C. The results also revealed that dried apple slices were able to provide prominent protection to the cells in acidic gastric juice.
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In the background of increasing research interest in food fermentations involving beneficial culture, the focus present study was to evaluate the ability of a single desirable bacterial culture to impart multiple benefits to the product being prepared therein. The intracellular α-d-galactosidase elaborated by Lactobacillus plantarum MTCC 5422 in formulated soy whey broth for Lactobacillus on partial purification exhibited an activity of 1.2 U/mL with specific activity of 12.0 U/mg and a threefold purification. The partially purified enzyme had a molecular mass of 84 kDa, pH and temperature optima of 5.8 and 45 °C, respectively, and enzyme stability of 30 min at 55 °C. Selected legume flours treated (2 h at 45 °C) with partially purified α-d-galactosidase resulted in lowering raffinose and stachyose levels in the range of 50–74 and 66–85 %, respectively. Soy-based fermented probiotic curd prepared with L. plantarum MTCC 5422 from α-d-galactosidase treated soy protein isolate showed almost 99 % decrease in levels of raffinose and stachyose. In the final product, the probiotic culture of L. plantarum MTCC 5422 reached viable population of 9.4 log10 CFU/g and elaborated short chain fatty acids such as acetic, butyric and propionic acids in appreciable quantity. The product was devoid of any contaminating foodborne pathogenic bacterial species. At the same time, the product had appropriate levels of pH, acidity and antibacterial activity against Bacillus cereus. The innovative approach was the lowering of raffinose and stachyose by α-d-galactosidase of L. plantarum MTCC 5422 and elaboration of probiotic attributes by the same culture in the final product.
The effectiveness as protective culture of the probiotic Lactobacillus rhamonosus GG (L. rham. GG) against Salmonella and Listeria monocytogenes on minimally-processed apples throughout storage as well as its effect on apple quality and natural microflora was evaluated. Survival to subsequent exposure to gastric stress was also reported. Apples were cut into wedges and dipped in a solution containing Salmonella and L monocytogenes (10(5) cfu mL(-1)) and/or L. rham. GG (10(8) cfu mL(-1)). Apple wedges were packed and stored at 5 and 10 degrees C. Periodically, microbial population, bacterial survival to gastric stress and quality of apple wedges were evaluated. Although Salmonella was not affected by co-inoculation with L. rham. GG, L monocytogenes population was 1-log units lower in the presence of L rham. CC. L. rham. GG population maintained over recommended levels for probiotic action (10(6) cfu g(-1)) along storage, however, viable cells after gastric stress were only above this level during the first 14 days. Pathogen survival after gastric stress was <1% after 7 days at 5 degrees C. Moreover, apple wedges quality was not affected by L. rham. GC addition. Thus, L. rham. GG could be a suitable probiotic for minimally-processed apples capable to reduce L monocytogenes growth; nevertheless shelf life should not be higher to 14 days to guarantee the probiotic effect.
The aim of this study was to evaluate the effect of microencapsulation (ME) in alginate beads on the viability of Bifidobacterium longum 15708 in terms of their tolerance to freezing, storage in a frozen state, cheddar cheese manufacturing and storage as well as to a simulated gastro-intestinal environment. Two ME methods namely i) droplet extrusion method (ADE) and ii) emulsion method, involving two polymers (native (NA) and palmitoylated alginate (PA)) were compared. Results showed that ADE maintained higher viability of B. longum after 24 h freezing at −80 °C with no viability loss as compared to the emulsion process and free cells which lost approximately 0.8 and 1.5 log CFU/mL respectively. However, during a 4 weeks storage period at −80 °C, no significant difference (P > 0.05) was observed in the survival of free and immobilized B. longum, with no loss of viability. Cheddar cheeses supplemented with B. longum culture were prepared and analysed during storage at 4 °C. After 21 days of storage, Cheddar cheese containing encapsulated B. longum in NA and PA polymers produced with the emulsion process showed a good survival with 2 log CFU/mL reduction after 21 days, as compared to ADE-encapsulated B. longum and free cells with 3 and 4 log CFU/mL reductions respectively. The immobilized bacteria in both polymers were also more resistant than free cells to simulated gastric and intestinal environments by a factor of 30.
This work deals with the design and formulation of gelatin edible coatings and films of incorporated with probiotic bacteria Lactobacillus acidophilus and Bifidobacterium bifidum. A gelatin coating incorporated with B. bifidum was applied to fish to assess its effect during chilled storage. B. bifidum remained viable during the storage and H2S-producing microorganisms were reduced in 2 log cycles. In a further experiment, fish coated with gelatin films incorporated with bifidobacteria was treated with a high pressure (200 MPa/10 min/20 °C). At the end of the storage, a reduction of total viable counts (< 2 log cycles) were obtained. Moreover, the combined treatments drastically reduced the content of volatile bases and lowered the pH by more than 1 unit. Furthermore, the application of gelatin edible packaging incorporated with bacteria can be promising for fish preservation, especially when combined with other technologies such as a high-pressure.Industrial relevanceGelatin edible coatings and films could act as a proper matrix to incorporate lactic acid bacteria to some foods. The application of those edible packaging confer stability during storage of chilled fish, especially in combination with high pressure treatment. These novel packaging was promising for fish preservation.