1341

Review

Received: 21 December 2010

Revised: 4 February 2011

Accepted: 4 February 2011

Published online in Wiley Online Library: 28 March 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4367

Probiotics and prebiotics – perspectives
and challenges

Ivonne Figueroa-Gonz ´alez, Guillermo Quijano,

∗

Gerardo Ram´ırez

and Alma Cruz-Guerrero

Abstract

Owing to their health benefits, probiotics and prebiotics are nowadays widely used in yogurts and fermented milks, which
are leader products of functional foods worldwide. The world market for functional foods has grown rapidly in the last three
decades, with an estimated size in 2003 of ca US$33 billion, while the European market estimation exceeded US$2 billion in
the same year. However, the production of probiotics and prebiotics at industrial scale faces several challenges, including the
search for economical and abundant raw materials for prebiotic production, the low-cost production of probiotics and the
improvement of probiotic viability after storage or during the manufacturing process of the functional food. In this review,
functional foods based on probiotics and prebiotics are introduced as a key biotechnological field with tremendous potential
for innovation. A concise state of the art addressing the fundamentals and challenges for the development of new probiotic-
and prebiotic-based foods is presented, the niches for future research being clearly identified and discussed.

c

2011 Society of Chemical Industry

Keywords: functional food; probiotics; prebiotics; synbiotics

INTRODUCTION

Nowadays, the strong relationship between diet and health is
well accepted. Although the primary role of diet is to provide
nutrients to fulfil metabolic requirements, the use of foods to
improve health and the state of wellbeing is an idea increasingly
accepted by society in the last three decades.

1 – 3

The change

in the way of conceiving foods has led to the introduction of
the concept of functional foods. Besides exhibiting an adequate
nutritional value, a food can be considered as functional if it
beneficially affects one or more target functions in the body
in a way that is relevant to either the state of wellbeing and
health or the reduction of the risk of a disease.

4

‘Functional

foods’ as a marketing term was initiated in Japan in the late
1980s. However, the world market has grown rapidly, with an
estimated size in 2003 of ca US$33 billion, while the European
market estimation exceeded US$2 billion in the same year.

5

The

functionality of functional foods is based on bioactive components,
often contained naturally in the product but usually requiring
the addition of a specific ingredient in order to optimise the
beneficial properties.

1

Today, functional food ingredients include

probiotics, prebiotics, vitamins and minerals, which are used in
fermented milks and yogurts, sports drinks, baby foods, sugar-free
confectionery and chewing gum.

6,7

Probiotics and prebiotics are

fundamental ingredients of fermented milks and yogurts, which
account for the most important fraction of the overall market
for functional foods. Therefore most of the available research on
functional foods has focused on probiotics and prebiotics.

5,6,8,9

This article constitutes a state-of-the-art review of probiotics and
prebiotics from the fundamentals to the concept of synbiotics and
challenges for the development of new probiotic- and prebiotic-
based foods. Finally, the key research niches in the field are
identified and discussed.

PROBIOTICS

Several definitions of probiotics can be found in the literature.
Hence, a decade ago, probiotics were considered as those viable
micro-organisms that exhibit a beneficial effect on the health of
the host by improving its intestinal microbial balance.

10

However,

a more recent and comprehensive definition is provided by Fric:

11

‘probiotics are non-pathogenic micro-organisms, mostly of human
origin, which confer a health benefit on the host and enable to
prevent or improve some diseases when administered in adequate
amounts’. The initial notion of probiotic micro-organisms can be
traced to a century ago when the Nobel Laureate Ilya Metchnikoff
noticed that the long healthy life of Bulgarian peasants resulted
from the consumption of fermented milk products.

2

Metchnikoff

suggested that, when consumed, the fermenting bacilli positively
influenced the microbiota of the gut, since these micro-organisms
were theoretically able to be established in the intestinal tract,
decreasing the pathogenic bacterial population.

12

Nevertheless,

later investigations revealed that the micro-organisms involved
in milk fermentation are not able to be established in the
gut.

13,14

Therefore the original idea of Metchnikoff regarding the

mechanism by which milk-fermenting micro-organisms provide
health benefits to the host was fundamentally wrong.

Nowadays, the most widely used probiotics include lactobacilli,

bifidobacteria and some non-pathogenic strains. These probiotic

∗

Correspondence to: Guillermo Quijano, Departamento de Biotecnolog´ıa,
Universidad Aut´

onoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No.

186 Col. Vicentina, CP 09340, Distrito Federal, Mexico.
E-mail: [e-mail]

Departamento de Biotecnolog´ıa, Universidad Aut´

onoma Metropolitana-

Iztapalapa, Av. San Rafael Atlixco No. 186 Col. Vicentina, CP 09340, Distrito
Federal, Mexico

J Sci Food Agric 2011; 91: 1341–1348

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2011 Society of Chemical Industry

1342

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I Figueroa-Gonz ´alez et al.

Table 1.

Key probiotic micro-organisms reported in the literature

Micro-organism

Effect on human health

Reference

Lactic acid bacteria

Lactobacillus rhamnosus GG

Reduces the intestinal permeability defects caused by exposure

to cow’s milk or rotavirus infection. May shorten the course of
rotavirus infection causing diarrhoea, traveller’s diarrhoea and
antibiotic-associated diarrhoea

10

Lactobacillus casei

Reduces the severity and duration of diarrhoea. Stimulates the

immune system of the gut, alleviates the symptoms of Crohn’s
disease and possesses strong antimicrobial properties

10,17,18

Lactobacillus casei Shirota

Prevents diarrhoea caused by viruses or bacteria. Has the

strongest human health efficacy with respect to management
of lactose malabsorption, rotaviral diarrhoea,
antibiotic-associated diarrhoea and Clostridium difficile
diarrhoea. Has a preventive effect on the recurrence rate of
superficial bladder cancer after surgery

18,19

Lactobacillus acidophilus

Secretes lactic acid that lowers the pH of the intestinal content

and helps to inhibit the development of invasive pathogens
such as Salmonella spp. or strains of Escherichia coli. Increases
antibody responses and seroconversion rates. Lowers serum
cholesterol levels

17,18,20

Lactobacillus johnsonii

May reduce density of Helicobacter pylori and inflammation as

well as gastritis activity

18

Lactobacillus plantarum

Produces short-chain fatty acids that inhibit the generation of

carcinogenic products by reducing enzyme activities

18

Bifidobacteria

Bifidobacterium breve

Activates the humoral immune system by augmenting

anti-rotavirus IgA production or anti-influenza virus

10

Bifidobacterium bifidum

May successfully compete for space and nutrients against

pathogenic or putrefactive bacteria. Reduces the incidence of
diarrhoea and increases antibody responses and
seroconversion rates

17,20

Bifidobacterium infantis

Prevents diarrhoea and constipation

11

Bifidobacterium animalis

Normalises the intestinal motility of obstipated subjects. Reduces

the risk of acute diarrhoea in children and adults

2,19

Yeasts

Saccharomyces cerevisiae Boulardii

Prevents traveller’s diarrhoea and the development of colitis and

enterocolitis of pathogenic origin. Reduces the risk and
duration of antibiotic-associated diarrhoea

11,21

cultures include Lactobacillus rhamnosus GG, Saccharomyces
cerevisiae Boulardii, Lactobacillus casei Shirota and Bifidobacterium
animalis, which are by far the most studied probiotics with
proven human health efficacy.

2,7,11,15

A brief summary of key

probiotics reported in the literature and their effect on health
is provided in Table 1. Most probiotics belong to the genera
Lactobacillus and Bifidobacterium, while S. cerevisiae Boulardii is
the only yeast with proven probiotic characteristics. According
to Saier and Mansour,

16

probiotics possess three mechanisms of

promoting human health: (i) providing end-products of anaerobic
fermentation of carbohydrates such as organic acids that can
be absorbed by the host, these end-products being able to
influence human mood, energy level and even cognitive abilities;
(ii) successfully competing with pathogens; (iii) stimulating host
immune responses by producing specific polysaccharides.

It is important to note that health benefits provided by probiotics

are strain-specific and not species- or genus-specific. Therefore no
probiotic strain will provide all proposed benefits, not even strains
of the same species. Likewise, not all strains of the same species
will be effective against defined health conditions.

18

Nonetheless,

it is observed that a common benefit of most probiotics listed in
Table 1 is their control of diarrhoea, this being associated with the
competitive exclusion of pathogens.

2

Thus the ability to survive

through the upper gastrointestinal tract and colonise the intestine
is a key criterion to consider a micro-organism as probiotic.

16,17

PREBIOTICS

The concept of prebiotics has also evolved significantly over time. A
prebiotic was first defined as a non-digestible food ingredient that
beneficially affects the host by selectively stimulating the growth
and/or activity of one or a limited number of bacteria in the colon,
thus improving host health.

22

However, recent literature does not

restrict the colon as the only action site and defines a prebiotic as
a selectively fermented ingredient that allows specific changes in
the composition and/or activity of the gastrointestinal microbiota
that confer benefits upon host wellbeing and health.

23

The key

concept associated with both these definitions is that prebiotics
have a selective effect on the microbiota that results in improved
health of the host.

24

An ingredient must fulfil three fundamental

aspects in order to be considered as a prebiotic: (i) resistance
to digestion; (ii) fermentation by the large intestinal microbiota;
(iii) a selective effect on the microbiota that has associated health-
promoting effects.

25

Therefore, in order to be effective, prebiotics

need to reach the large bowel with their chemical and structural
properties essentially unchanged to further selectively stimulate
the microbiota. Most prebiotics are short-chain carbohydrates with
a degree of polymerisation of 2 or more, which are not susceptible
to digestion by pancreatic and brush border enzymes.

24

A brief

summary of the main candidates for prebiotic status is provided
in Table 2. Prebiotic oligosaccharides may be manufactured by
extraction from plant materials, microbial/enzymatic synthesis
and enzymatic hydrolysis of polysaccharides. Various prebiotics

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Probiotics and prebiotics – perspectives and challenges

www.soci.org

Table 2.

Main candidates for prebiotic status reported in the literature

Carbohydrate

Chemical structure

Method of manufacture

Degree of polymerisation

Reference

Inulin

β(2-1)-Fructans

Extraction from chicory root and Agave

tequilana

2–65

25,27,31,32

Fructo-oligosaccharides

β(2-1)-Fructans

Transfructosylation from sucrose or

hydrolysis of chicory inulin

2–10

25,33

Galacto-oligosaccharides

Galactose oligomers and

some
glucose/lactose/galactose
units

Produced from lactose by

β-galactosidase

2–5

25,34 – 36

Soya-oligosaccharides

Mixture of raffinose and

stachyose

Extracted from soya bean whey

3–4

25,33

Xylo-oligosaccharides

β(1–4)-Linked xylose

Enzymatic hydrolysis of xylan. Enzyme

treatments of native lignocellulosic
materials. Hydrolytic degradation of
xylan by steam, water or dilute
solutions of mineral acids

2–4

25,37

Isomalto-oligosaccharides

α(1–4)-Glucose and

branched α(1–6)-glucose

Microbial or enzymatic

transgalactosylation of maltose.
Enzymatic synthesis from sucrose

2–8

25,38

Pyrodextrins

Mixture of

glucose-containing
oligosaccharides

Pyrolysis of potato or maize starch

Various

25

are produced at industrial scale and are widely available in the
market.

26

As a matter of fact, prebiotics have been reported as

cheap to manufacture.

27

This perception might be inspired by

the fact that prebiotics are obtained from relatively low-cost raw
materials.

28

However, most prebiotics are obtained by means of

enzymatic processes and thus synthesis and further purification is
not necessarily a cheap process. In fact, several investigations on
novel aqueous/organic media for enzymatic synthesis have been
explored in order to improve the yield of specific prebiotics.

29,30

Aiming to develop a quantitative tool to compare the effect

of prebiotics on the growth of probiotic bacteria, Palframan
et al.

39

proposed an equation in which a prebiotic index (PI) is

introduced. The PI score is based on the selective stimulation
of probiotic bacteria (bifidobacteria and lactobacilli) over other
micro-organisms (bacteroides and clostridia) due to addition of a
prebiotic according to the following equation:

PI

= Bif/Total + Lac/Total − Bac/Total − Clos/Total

(1)

where Bif, Lac, Bac, Clos and Total represent the concentration
ratios of bifidobacteria, lactobacilli, bacteroides, clostridia and total
bacteria between sampling time and at inoculation respectively.
A negative PI score indicates that the prebiotic addition did not
selectively stimulate the growth of probiotic bacteria, while a
positive PI value indicates that the tested prebiotic was able
to specifically stimulate probiotic bacteria under the working
conditions. Unfortunately, this equation has two important
drawbacks: (i) the amount of prebiotic consumed is not considered
and thus a fair comparative analysis among several prebiotics is
restricted to studies with similar prebiotic uptake; (ii) the PI defined
in Eqn (1) is useful only for experiments in vitro where the total
concentration of probiotics and pathogens is well known. In the
case of in vivo experiments the total bacterial population as well
as the prebiotic uptake in the host is fundamentally unknown.
Hence the increase in probiotic population due to prebiotic
consumption is only inferred through the content of probiotics in
faecal matter.

23

Based on these key differences between in vitro

and in vivo experiments, it is not fair to compare their PI scores.
Recently, Roberfroid

23

proposed a new PI for in vivo experiments

based on the generation of new probiotic bacteria per gram of
prebiotic ingested:

PI

= [(CFU/g

faeces

)

final time

− (CFU/g

faeces

)

initial time

]/Prebiotic dose

(2)

where CFU denotes colony-forming units and the Prebiotic dose
refers to the number of grams of prebiotic ingested per day by the
host. The advantage of calculating the PI by means of Eqn (2) is that
the effect on probiotic growth is based on prebiotic concentration.
Therefore a fair comparative analysis can be performed even for
studies with different prebiotic doses. However, the author did
not find a correlation between the PI values and the amount of
prebiotic ingested. This might have been due to the fact that
the ingested prebiotic is not necessarily the amount of prebiotic
that reaches and is consumed in the large intestine by probiotic
bacteria.

SYNBIOTICS

It is nowadays common to find probiotic foods (e.g. yogurts and
fermented milks) with added prebiotics. Such a combination, in
which the concentration of prebiotic is typically below 10 g kg

−1

,

is known as a ‘synbiotic’.

19

Synbiotics have been considered as

a very promising area for the development of new functional
foods. In fact, the potential synergy between probiotics and
prebiotics has been considered as ‘obvious’.

40

Synbiotics have

also been defined as mixtures of probiotics and prebiotics
that beneficially affect the host by improving the survival
and implantation of live microbial dietary supplements in its
gastrointestinal tract.

41

In vitro simulations of the gastrointestinal

tract constitute a common experimental framework to investigate
synbiotics (Table 3). One of the main benefits of synbiotics is the
increased persistence of probiotics in the gastrointestinal tract.

42

Therefore the reported in vitro experiments are mainly focused
on elucidating (i) if probiotics might metabolise the prebiotic

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I Figueroa-Gonz ´alez et al.

Table 3.

Examples of synbiotics reported in the literature

Synbiotic

Probiotic

Prebiotic

Type of experiment

Reference

Lactobacillus casei strain Shirota

Oligomate 55



In vitro

45

Bifidobacterium longum

Oligofructose

In vivo (rats)

43

Bifidobacterium lactis Lafti



B94

Resistant starch

In vitro

40

Bifidobacterium breve strain Yakult

Galacto-oligosaccharides

In vivo (mice)

46

Lactobacillus gasseri

Inulin and unspecified oligosaccharides

In vivo (humans)

44

Lactobacillus acidophilus ATCC 4962

Manitol, fructo-oligosaccharides and inulin

In vitro

47

Lactobacillus sakei JCM

Fructo-oligosaccharides and trehalose

In vitro

48

Lactobacillus plantarum and L. acidophilus

Xylo- and fructo-oligosaccharides

In vitro

49

component under simulated conditions of the gastrointestinal
tract, (ii) if the prebiotic component may be metabolised by
pathogenic bacteria and (iii) if the synbiotic is able to inhibit
or decrease the growth of pathogenic bacteria with respect to
a control without synbiotic. Despite the relevant information
obtained from in vitro tests, authors working with this kind of
experiment clearly recognise that further in vivo trials are necessary
to corroborate the effectiveness of synbiotics.

40

In this context,

de Vrese and Schrezenmeir

19

highlighted that synbiotics might

improve the survival and implantation of probiotics only in the
case of a true synergistic mutual reinforcement between the
prebiotic and probiotic components. Lately, it is not clear whether
synbiotics will result in elevated levels of probiotic bacteria
owing to the prebiotic component.

42

For instance, a synbiotic

based on Bifidobacterium longum and oligofructose stimulated
the proliferation of faecal bifidobacteria by 1.4 log(CFU g

−1

)

in Wistar rats, while an increase in faecal bifidobacteria of 1.6
log(CFU g

−1

) was obtained by administering only oligofructose.

43

Moreover, Morelli et al.

44

assessed the effect of a synbiotic based

on Lactobacillus gasseri (strain B21090), inulin and unspecified
oligosaccharides on the growth of probiotic bacteria. Although
these authors found that the synbiotic was effective in increasing
the population of faecal probiotic bacteria in human volunteers,
the stimulation of probiotic bacteria by synbiotic administration
was not compared with the stimulation obtained with the prebiotic
alone. Consequently, a comparative analysis of the potential
superior effectiveness of the synbiotic over the prebiotic by
itself cannot be made. Therefore convincing evidence for the
real potential of synbiotics should come from more in vivo studies
comparing the performance of synbiotics with respect to their
components.

CHALLENGES FOR NEW PROBIOTIC-
AND PREBIOTIC-BASED FOODS

Owing to their perceived health benefits, probiotics and prebiotics
are now widely added to yogurts and fermented milks.

5,6,50

However, the production of probiotics and prebiotics at industrial
scale faces several challenges, including (i) the use of novel
techniques and economical sources for prebiotic production,
(ii) the low-cost production of probiotics and (iii) the improvement
of probiotic viability after storage, during the manufacturing
process of the functional food and during transit through the
stomach. These key challenges are addressed below.

Novel techniques and economical sources for prebiotic
production
Most oligosaccharides with prebiotic status are normally obtained
by enzymatic treatment of cheap raw materials such as sucrose,
lactose and plant derivatives (Table 2). The amount and nature of
the oligosaccharides formed depend upon several factors such as
the enzyme source, the concentration and nature of the substrate
and the reaction conditions.

32

Nevertheless, the current processes

to obtain oligosaccharides have very low yields, thus increasing
the production cost.

29,51

Panesar et al.

34

noted that the yield

of oligosaccharides can be increased by decreasing the water
content in the reaction medium. In this regard, del Val and Otero

29

studied the enzymatic synthesis of galacto-oligosaccharides from
lactose in aqueous/polyethylene glycol media. They found that a
decrease in water content increased not only the overall yield of
galacto-oligosaccharides but also the selective production of 6

-

galactosyl lactose. In addition, Cruz-Guerrero et al.

30

investigated

the synthesis of galacto-oligosaccharides from lactose using a
hyperthermophilic β-glycosidase in aqueous/acetone medium.
They observed that the oligosaccharide yields were strongly
affected by the water activity (a

w

), the maximum yield being

obtained at an a

w

of 0.57 and an initial lactose concentration

of 8 g L

−1

. Therefore the enzymatic production of prebiotics in

organic media represents an interesting research field to improve
the yield of prebiotics over traditional synthesis in aqueous media.
The reviewed literature already focuses on such research to
establish optimum aqueous/organic volume ratios or a

w

/substrate

concentration ratios when the synthesis medium consists mainly
of an organic solvent.

The production of non-dairy oligosaccharides is another

interesting option for low-cost prebiotic production at industrial
scale. In recent years, cereals have been investigated regarding
their potential as sources of prebiotics. Charalampopoulos
et al.

52

concluded that at least two types of naturally occurring

oligosaccharides can be found in cereal grains, namely galactosyl
derivatives of sucrose, stachyose and raffinose and fructosyl
derivatives of sucrose. On the other hand, cereal grains are rich
in dietary fibre, which encompasses a heterogeneous range of
complex polysaccharides that are not substantially digested in the
small intestine and pass through to the colon. Thus dietary fibre
constitutes a potential source of prebiotics from cereals.

53

The

dietary fibre content in grains such as rice, rye and corn can reach
values as high as 150–190 g kg

−1

dry matter (Table 4). β-Glucans,

xylans, xylo-oligosaccharides and arabinoxylans are the main
constituents of dietary fibre. Crittenden et al.

53

reported that many

Bifidobacterium species and Lactobacillus brevis were able to grow

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Probiotics and prebiotics – perspectives and challenges

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Table 4.

Total dietary fibre content in several cereal grains

Cereal

Total dietary fibre (g kg

−1

dry matter)

Reference

Wheat

121

55

Rye

161

55

Barley

100

52

Sorghum

107

52

Rice

192

55

Corn

150

52

Oat

140

52

at high yields using xylo-oligosaccharides. They also observed that
B. longum strains were able to grow using arabinoxylan as the
sole carbon source. In addition, hydrolysates of β-glucans have
been reported to stimulate the growth of several Bifidobacterium
strains and L. rhamnosus GG.

52

Recently, Moura et al.

54

selectively

produced oligosaccharides with degrees of polymerisation of 3–4
and 5–6 by means of autohydrolysis techniques using corn cobs
as the raw substrate. Moreover, the obtained oligosaccharides
promoted the growth of both Bifidobacterium and Lactobacillus
species to a similar extent to commercial xylo-oligosaccharides.

Starch is a substantial component of many cereal grains such as

corn, wheat, rice and oat (785, 650, 820 and 636 g kg

−1

dry matter

respectively).

56

Although resistant starch is found naturally in

cereal grains, it is frequently destroyed when subjected to modern
food processes. Industrial methods for manufacturing resistant
starch include partial acid hydrolysis, hydrothermal treatment,
heating, retrogradation, extrusion cooking, chemical modification
and repolymerisation.

52

As in the case of dietary fibre, studies

in animals and human volunteers have shown that a fraction
of the total starch is not digested in the small intestine and
passes through to the colon. This fraction is termed resistant
starch and has been reported as prebiotic.

57

Crittenden et al.

40

reported that many Bifidobacterium strains such as B. adolescentis,
B. bifidum, B. breve, B. infantis, B. lactis and B. longum were able
to hydrolyse resistant starch. However, only B. lactis (strain Lafti



B94) maintained its viability under conditions similar to those
encountered during passage through the gastrointestinal tract.
The authors finally proposed a synbiotic yogurt based on resistant
starch and B. lactis. On the other hand, Topping et al.

57

concluded

that evidence regarding resistant starch as prebiotic is still limited,
since much of the experimental work has been done in animals,
the available investigations in humans being of relatively short
duration. However, the authors recognised that there is a great
deal of promise, and further research is necessary to assess the
potential of resistant starch as a prebiotic in humans.

Low-cost production of probiotics
Low-cost production of concentrated cultures of probiotics is a
key challenge to satisfy the increasing demand for probiotics in
the market.

58

Probiotic micro-organisms are normally difficult to

grow as they lack biosynthetic capacity for most vitamins and
amino acids, so culture media for probiotic production must
be supplemented with both amino acids and vitamins.

59,60

The

reduction of production costs at industrial scale might start with
the use of some agro-industrial residual effluents as cultivation
media for probiotics. In this regard, effluents from fruit and
vegetable processing usually contain high amounts of proteins,
carbohydrates, lipids and vitamins. In the case of effluents from

cereal processing, mono-, di- and oligosaccharides can also be
found.

61

A clear example of this kind of effluent is the wastewater

from industrial corn processing. According to Guti ´errez-Uribe
et al.,

62

the wastewater from the manufacturing process of

commercial fresh masa and dry masa flour (also called nejayote)
contains a high load of corn solids. Approximately 50% of these
solids are suspended and contain about 64, 20 and 1.4% non-starch
polysaccharides, starch and protein respectively. The remaining
50% consist of proteins, sugars, vitamins and phytochemicals
rich in phenolics and carotenoids. Other studies revealed that
nejayote contains polysaccharides of arabinose, xylose, glucose,
galactose and

D

-glucuronic acid and approximately 23% crude

fibre (whose dietary fibre fraction may stimulate the growth
of probiotics).

52,53,63 – 65

Therefore agro-industrial effluents such

as the wastewater from cereal processing may be an abundant
source of prebiotic compounds. Efforts must be made to determine
the feasibility of using these effluents either partially or totally in
culture media formulation.

Improving viability of probiotics
The quality of probiotic micro-organisms relies greatly on their
viability, which is a fundamental fulfilment to reach and colonise
the human large intestine.

66,67

Probiotics must retain their viability

during three critical stages: (i) storage; (ii) manufacturing process
of the functional food; (iii) transit through the stomach and small
intestine. Therefore the viability of probiotics is an issue of vital
importance from both an economic and a technological viewpoint.
In this regard, Ratnakomala and Widyastuti

68

observed that storage

of a probiotic by freezing at

−40

◦

C decreased its viability from

1.8

× 10

15

to 1.6

× 10

10

CFU mL

−1

, while a freeze-drying pre-

treatment followed by storage at 4

◦

C decreased its viability from

8.9

× 10

14

to 2.4

× 10

9

CFU mL

−1

. Likewise, Lahtinen et al.

66

noted

that probiotics might become dormant during storage. They
observed that Bifidobacterium strains can be completely dormant
after storage at 4

◦

C for 25 days in an oat-in-water suspension. Such

results clearly indicate that storage may significantly decrease the
viability of probiotics. Freeze-drying in food matrices has been
proposed to improve probiotic viability during storage and transit
through the stomach and small intestine. For instance, Saarela
et al.

69

reported that freeze-drying in a matrix of low-fat milk or

fruit juice significantly improved the viability of a Bifidobacterium
strain. They observed that viability in the juice matrix was higher
than in low-fat milk during storage, while tolerance to acid and
bile was better in cells with the low-fat milk matrix after storage.
The authors concluded that, when choosing a food matrix as
cryoprotectant for a probiotic, viability should not be the only
parameter to be evaluated but also the ability to resist adverse
conditions after storage. Therefore research on low-cost food
matrices as cryoprotectants to improve probiotic viability after
storage is a relevant field that is worthy of further investigation.

On the other hand, microencapsulation is a promising technique

recently studied to improve the viability of probiotic micro-
organisms. Microencapsulation can be defined as a technology
of packaging solids, liquids or gases in miniature, sealed capsules
that can release their contents at controlled rates under the
influence of specific conditions.

70

It has been reported that

microencapsulation protects probiotics during storage and during
the manufacturing process of a determined functional food,
improved viability against heat stress and acidic conditions
also being observed.

70,71

Microencapsulation techniques used

so far to protect probiotic micro-organisms include spray-drying,
spray-congealing, fluidised bed coating/air suspension, extrusion,

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I Figueroa-Gonz ´alez et al.

Table 5.

Some examples of coating materials used in probiotic

microencapsulation

Coating material

Probiotic

Reference

Alginate/resistant starch

Lactobacillus

acidophilus and
Bifidobacterium
spp.

71

Whey protein

Lactobacillus

rhamnosus

71

Whey protein

Bifidobacterium

longum

70

Cellulose acetate phthalate

Bifidobacterium

pseudolongum

70

Alginate/sodium lauryl

sulfate

Lactobacillus

delbrueckii

70

Carrageenan/locust bean

gum

Lactobacillus casei

70

Alginate

Lactobacillus

acidophilus

70

Canola vegetable

oil/caseinate/fructo-
oligosaccharides/resistant
starch (or glucose syrup)

Bifidobacterium

infantis

72

Alginate/gellan gum/fructo-

oligosaccharides/peptides

Bifidobacterium

bifidum

48

coacervation/phase separation and electrostatic methods. A
detailed description of these microencapsulation technologies
is given by Anal and Singh.

70

The common material of all

microencapsulation techniques is the coating material in which
the probiotic is encapsulated. Common coating materials include
water-soluble/insoluble polymers, waxes, fatty acids and lipids.
Table 5 shows some of the most common probiotics and coating
materials used in probiotic microencapsulation. Prebiotics such
as fructo-oligosaccharides or resistant starch may be part of
the coating formulation. For instance, Chen et al.

48

included

fructo-oligosaccharides in the coating formulation for B. bifidum
microencapsulation. They observed that a formulation including
prebiotics, gellan gum and peptides significantly improved
probiotic viability with respect to microcapsules made from
alginate as the sole coating material. Likewise, Crittenden et al.

72

included prebiotics in the coating formulation for B. infantis
microencapsulation. They found that a combination of canola
vegetable oil, caseinate, fructo-oligosaccharides and resistant
starch (or glucose syrup) improved the viability of the prebiotics
by ten orders of magnitude relative to free cells after storage
for 5 weeks in an open container at 25

◦

C and 50% relative

humidity. Therefore the investigation of new coating materials and
microencapsulation techniques also constitutes a key research
niche. Available investigations seem to direct efforts towards
research on cheap and abundant coating materials as well as
optimisation of the coating material formulation (e.g. optimum
prebiotic percentages).

CONCLUSIONS

In brief, functional foods based on probiotics and prebiotics consti-
tute a very important biotechnological field with tremendous po-
tential for innovation. We herein identified three main challenges
for the industrial production of probiotic- and prebiotic-based
functional foods: (i) the improvement of production techniques

for prebiotic synthesis; (ii) the reduction of probiotic production
costs; (iii) the enhancement of probiotic viability during storage,
during the manufacturing process of the functional food and dur-
ing transit through the upper gastrointestinal tract. The strategies
to overcome such challenges must come from a multidisciplinary
approach. The alternatives herein reviewed include the following.

• Production of prebiotics in two-liquid-phase systems by adding

an organic solvent that increases the yield of prebiotics. Such
multiphase systems may also increase the yield of prebiotics
with specific degrees of polymerisation.

• Production of oligosaccharides from cheap and non-dairy raw

materials, where cereals seem to be the most interesting
option as they have naturally occurring oligosaccharides such
as galactosyl derivatives of sucrose, stachyose and raffinose as
well as fructosyl derivatives of sucrose. Moreover, cereal grains
are rich in dietary fibre and starch, the latter being able to be
modified to yield resistant starch.

• Probiotic production from agro-industrial effluents, where

corn-processing effluents appear as a very interesting option
based on their high concentrations of non-starch polysaccha-
rides, starch, protein, sugars, vitamins, phytochemicals rich in
phenolics and carotenoids as well as polysaccharides of ara-
binose, xylose, glucose, galactose and

D

-glucuronic acid. Thus

this kind of effluent may be totally or partially used for low-cost
culture media formulation.

• The viability of probiotics may be improved by using cheap

food matrices or by means of microencapsulation techniques.
Hence research on matrices such as milk or fruit juice could
potentially improve the viability of probiotics, as they act
as cryoprotectants during the freeze-drying process. On the
other hand, microencapsulation was identified as a promising
technique that protects probiotics during storage, during the
manufacturing process of a determined functional food and
during transit through the upper gastrointestinal tract.

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