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PROBIOTICS IN FRUITS AND VEGETABLES: TECHNOLOGICAL AND SENSORY ASPECTS

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Abstract

The increased consumer awareness of the relationship between diet and health, corresponds to a greater demand for food promoters of a general state of well-being, such as the functional foods. Among the functional foods, foods with probiotics occupy a large market share. The probiotics are generally added to milk products (yogurt, fermented milks and cheeses), but the consumption of these products is excluded for individuals lactose intolerant, vegans and for low-cholesterol diets. The development of non-dairy products includes the use of fruits and vegetables as matrices to which add beneficial microorganisms. Fruits and vegetables are also a natural source of prebiotics. In this review, the presence of probiotics in fruit and vegetables is discussed, with a focus on the effect of the addition of these microorganisms on the technological and sensory aspects.
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189
10
Probiotics and Prebiotics in Fruits and
Vegetables: Technological and Sensory Aspects
Fernanda Galgano, Nicola Condelli, Marisa Carmela Caruso,
Maria Antonietta Colangelo, and Fabio Favati
10.1 Intestinal Microbiota and Probiotic Microorganisms
It has been known for a long time that diet can have a positive or negative effect upon human health. In
industrialized countries, a diet based on too much fat is the cause of diseases, such as hypertension and
obesity. On the contrary, in developing countries, a poor diet causes serious nutritional deciencies and
death. Therefore, in recent years, there has been an increasing interest in food nutrition, with a view to
safeguarding health. In fact, consumers are more aware about the link between food and health; this
explains the reason for an increasing demand for foods that promote good health, such as functional
foods (Peres et al., 2012).
The term functional food was used for the rst time in Japan in the mid-1980s (Chonan, 2011). In addi-
tion to making nutrients, functional foods have a positive inuence on the health of the host by helping
to reduce the risk of contracting certain diseases (Cencic and Chingwaru, 2010; Stringheta et al., 2007).
The European Commission Concerted Action on Functional Food Science (FUFOSE) has written a
document that states that a food can be considered functional if it has been satisfactorily demonstrated
that, in addition to its nutritional value, it performs one or more functions benecial to the body, thus pro-
ducing a state of well-being and reducing the risk of certain types of diseases (Evangelisti and Restani,
2011). The group of functional foods includes foods with probiotic microorganisms.
Although each area of our body is colonized by microorganisms, most of them lie in our gut. Gut
microbiota is the terminology used to describe the huge amount of microorganisms that colonize the
entire digestive tract (Evangelisti and Restani, 2011). Thus, the original microbiota creates an important
barrier between the external environment and the individual, avoiding a wide variety of disorders (Kosin
and Rakshit, 2006; Vertuani and Manfredini, 2001).
However, it is possible to ingest some selected bacteria, such as probiotics, which may benecially
affect the human gastrointestinal tract. The rst person to realize the positive effect of selected micro-
organisms on human health was Eli Metchnikoff. He suggested that “the dependence of the intestinal
microbes on the food makes it possible to adopt measures to modify the ora in our bodies and to replace
the harmful microbes by useful microbes” (Morelli, 2000).
CONTENTS
10.1 Intestinal Microbiota and Probiotic Microorganisms ...................................................................189
10.2 Prebiotics .......................................................................................................................................191
10.3 Fruits and Vegetables as a Source of Probiotic Microorganisms ................................................ 192
10.3.1 Probiotic Fermented Beverages ....................................................................................... 194
10.4 Microincapsulation ....................................................................................................................... 198
10.5 Challenges for Probiotic Foods Formulation ............................................................................... 200
10.6 Conclusions .................................................................................................................................. 201
References .............................................................................................................................................. 201
190 Benecial Microbes in Fermented and Functional Foods
Probiotics are live microorganisms that, when administered in adequate amounts, confer a health
benet on the host, inuencing positively the gut microbiota (Granato et al., 2010; Sloan, 2004; Ziemer
and Gibson, 1998). The term probiotic (meaning for life in Greek) includes a large range of microorgan-
isms, mainly bacteria, but also yeasts, although their effect on human health is strain specic (Burgain
et al., 2011). The main species of probiotic bacteria added to food are Lactobacillus and Bidobacterium
(Champagne et al., 2011; Saulnier et al., 2009).
In order to be tested for human probiotic use, a microorganism must have the following specic
characteristics:
It must be of human origin.
It must be nonpathogenic.
It must be resistant to degradation by gastric and pancreatic juices.
It must be able to adhere to the intestinal epithelium.
It must be able to colonize the gastrointestinal tract.
It must be a producer of antimicrobial substances.
It must be a modulator of the immune response (Morelli, 2000).
In addition, higher levels of viable microorganisms (1 × 107 CFU/g) are recommended in probiotic foods
for better efcacy in human organism (Ranadheera et al., 2010).
It is well known that probiotics are able to modify the intestinal microbiota by reducing the pH,
producing substances having antibacterial action, and stimulating the immune system (Agrawal, 2007;
Gerritsen et al., 2011; Zubillaga et al., 2001).
Microorganisms commonly used in probiotic foods are Lactobacillus species, such as L. acidophilus,
L. casei, L. reuteri, L. rhamnosus, L. johnsonii, and L. plantarum, and Bidobacterium species, such as
B. longum, B. breve, and B. lactis. Some scientic studies suggest that probiotics are able to treat Crohn’s
disease and ulcerative colitis (Kruis et al., 2004; Marteau et al., 2006; Prantera et al., 2002; Van Gossum
et al., 2007). They also play a protective role against enteric infections caused by Salmonella, Listeria
monocytogenes, and Clostridium difcile (Koninkx and Malago, 2008). Selected strains of lactobacilli,
L. casei GG and L. reuteri, as well as Saccharomyces boulardii, are effective for reducing the duration
of diarrhea associated with gastroenteritis (Bhadoria and Mahapatra, 2011).
L. rhamnosus GG (LGG), L. plantarum, and S. boulardii strains are most commonly used for
infections attributed to C. difcile (Marangoni, 2004). However, the intake of L. johnsonii is effective
for the inhibition and eradication of Helicobacter pylori (Sanchez et al., 2009), while Bidobacterium
animalis strain seems to have an effect in speeding intestinal transit and combat constipation (Sanchez
et al., 2009).
The concept of bioprotica derives from analyzing the characteristics that a probiotic must have and
its eld of action. One of the characteristics of a probiotic is that it must be of human origin. The fact
that microorganisms with probiotic characteristics are present not only in the intestine but also in other
areas of the body gave rise to the idea of using specic probiotic strains for the treatment of different
pathologies. The probiotic strains of the skin, for example, could be used to antagonize the growth and
proliferation of pathogenic fungi for the skin. Nonpathogenic strains of human derivation, resistant to
the acid pH of the gastric environment, could be used to combat the proliferation of H. pylori. According
to the selection area of the strain, the probiotics can be used for the treatment of specic disorders. The
term bioprotica was coined on the basis of these considerations.
Literally, the term bioprotic refers to the use of living bacteria (bio) for the treatment of a specic
sickness (pro stands for in favor of ) with a well-identied and precise pharmaceutical or nutraceutical
(tic) treatment (Di Pierro et al., 2013). Bioprotica means trying to select strains to be used in a specic
way; for example, a strain of the oral cavity of Streptococcus salivarius K12, which highly produces
bacteriocins and also able to colonize the area, has been isolated and tested for clinical evaluation in
the prevention of recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes in adults (Di
Pierro et al., 2013, Tagg, 2004). Moreover, the results of numerous studies on the metabolic prole and
toxicity tests have shown the S. salivarius K12 to be safe for human use (Burton et al., 2006).
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191Probiotics and Prebiotics in Fruits and Vegetables
10.2 Prebiotics
Prebiotic is a non-digestible substance of food origin which, when administered in adequate
amounts, is benecial to the consumer due to the selective promotion of growth and/or activity
of one or more bacteria already present in the gastrointestinal tract or taken together with the
prebiotic. (Hill et al., 2014)
Foods with prebiotics contain at least one nondigestible ingredient by humans, able to selectively promote
the growth and activity of benecial microbial species that colonize the intestine (Charalampopoulos
and Rastall, 2011; Gibson et al., 2004; Roberfroid, 2007).
Prebiotics must have some particular characteristics:
They must not be hydrolyzed nor absorbed in the upper digestive tract.
They must represent a selective substrate for one or more benecial bacterial species in the
colon stimulating their growth or activity.
They must be able to modify the intestinal microora of the colon promoting a healthy
composition.
Prebiotics are used only by benecial microorganisms that colonize the gut, without promoting the
growth and multiplication of pathogenic microorganisms, such as Escherichia coli and clostridia.
All the food components that, irrespectively of their chemical nature, are able to reach the colon with-
out any degradation, are not hydrolyzed in the upper digestive tract, stimulate the growth of microbial
species present in the gut, and promote a healthy composition of the intestinal microora can be consid-
ered to be prebiotics. Most prebiotics are nondigestible carbohydrates (nondigestible oligosaccharides or
bers), such as fructooligosaccharides (FOS) with bidogenic action, since these stimulate the growth of
bidobacteria (Evangelisti and Restani, 2011; Ziemer and Gibson, 1998).
Small amounts of FOS are contained in onions, garlic, artichokes, wheat, rye, asparagus, and bananas.
However, the food industry produces drinks, cookies, crackers, fermented milks, yogurts, and even
herbal teas with added prebiotic bers. From a technological point of view, the addition of prebiotics
does not require special production technologies, as these ingredients are not altered by air and heat
(Evangelisti and Restani, 2011). Even products for babies, such as weaning food and milk, often contain
added prebiotic components. Infant formula enriched with prebiotics is more similar to breast milk,
naturally rich in prebiotics. The prebiotic component of breast milk is of great importance, considering
that it is the third most abundant solid component and has a major role in protecting the baby from bacte-
rial infection. Prebiotics act by preventing the adhesion of pathogenic bacteria and preventing urinary
tract infections in the child. Breastfeeding is always recommended for both the child’s health and for
the well-being of the mother. However, for cases in which breastfeeding is not possible, it is necessary
to provide replacement formulations containing prebiotics to make them more similar to breast milk
(Vandenplas, 2002).
A controversial question regards the effect of the addition of prebiotics on the sensory characteristics
of the food. The incorporation of prebiotics in the food matrix has immediate effects on the avor and
texture of the food; therefore, these ingredients could replace the fat component that usually gives soft-
ness and creaminess to food (Ranadheera et al., 2010).
Hardi and Slanac (2000) have reported the effect of prebiotics on the stability of the pH and the post-
acidication of the fermented milks, showing that in a fermented milk the pH decreases more quickly
when inulin is added. On the other hand, Zhu (2004) has reported that the pH and acidity of yogurt
do not change after the addition of 4% of FOS. Inconsistent results make further studies and research
ne c essary.
There is synergy between probiotics and prebiotics, as in the colon probiotics use prebiotic compounds
as a source of energy. As a consequence, the activity of pathogenic microorganisms in the gut is further
reduced (Homayouni et al., 2008).
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192 Benecial Microbes in Fermented and Functional Foods
By a combination of probiotics and prebiotics, it is possible to formulate symbiotic foods. Symbiotic
foods contain a probiotic and a prebiotic component. Several studies (Gmeiner et al., 2000; Gomes and
Malcata, 1999; Roberfroid et al., 1998; Schaafsma et al., 1998) suggested that the consumption of sym-
biotic foods affects the human health more than the consumption of a food containing only probiotics or
prebiotics alone. The presence of probiotics and prebiotics in a single food improves probiotic viability
during storage of the product and during the passage through the gastrointestinal tract. In addition, the
symbiotic food allows an efcient implantation of probiotics in the colon due to the stimulating effect
of the latter on the growth and activity of the microorganisms (Roberfroid et al., 1998). Unfortunately,
the production of symbiotic foods results in increased costs for both the industry and the consumer. The
use of probiotic bacteria able to synthesize prebiotics might overcome this limitation. Some authors
reported that some bidobacteria strains are capable to synthesize galactooligosaccharides (Dumortier
et al., 1990; Van den Brook et al., 1999).
10.3 Fruits and Vegetables as a Source of Probiotic Microorganisms
The incorporation of probiotic strains in food matrices principally concerns dairy products, such as
fermented milks and cheeses (Garcia-Fontan et al., 2006; Hagen and Narvhus, 1999; Laroia and Martin,
1991; Lourens-Hattingh and Viljeon, 2001; Ong et al., 2006; Tharmaraj and Shah, 2004). However, other
foods have been studied as potential probiotic carriers, such as fermented sausages, coconut desserts,
ice cream, and chocolate mousse (Casale Aragon-Alegro et al., 2007; Homayouni et al., 2008; Lavasani
et al., 2011; Madureira et al., 2011).
Although fermented milk and yogurt with probiotic products are the most popular on the market, it
is necessary to propose viable alternatives to products that do not contain milk, both to meet the needs
of those people suffering from lactose intolerance or hypercholesterolemia and to meet the growing
demand for vegetarian products (Peres et al., 2012; Ranadheera et al., 2010). For this reason, there is an
increasing demand for vegetarian probiotic products (Ranadheera et al., 2010). Foods based on fruit and
vegetables, such as fruit and vegetable juices, represent a new potential carrier and source of probiotic
microorganisms (Nicolesco and Buruleanu, 2010; Nualkaekul and Charalampopoulos, 2011; Pereira
et al., 2011; Peres et al., 2012; Sheela and Suganya, 2012). Raw and fermented vegetables also represent
an excellent vehicle for probiotics due to their natural structure that allows the easy availability of use-
ful nutrients for microbial growth (Espírito-Santo et al., 2011; Peres et al., 2012; Soccol et al., 2010).
The tissues of plants contain intracellular spaces, pores, and capillaries. Although the cell walls of
plants are very resistant, the microorganisms are able to penetrate through the tissue injury (Brackett
and Splittstoesser, 2001). The microorganisms enter the pores, cracks, and lesions of the surface of the
fruits. Some operations such as peeling and cutting performed on minimally processed products can
favor the availability of nutrients, such as sugars, vitamins, and minerals needed for probiotic growth (de
Oliveira et al., 2011; Rößle et al., 2010; Soccol et al., 2010). Lactic acid bacteria isolated from the same
plant can be used as probiotics (Alzamora et al., 2005; Betoret et al., 2003; Renadheera et al., 2010).
Moreover, most fruits and vegetables contain prebiotic ingredients that promote the growth of benecial
microorganisms.
Some products, processed from fermented vegetables, are already widely consumed in some parts
of the world. An example is kinema, obtained by the fermentation of soybeans using Bacillus subtilis
and consumed by the inhabitants of Nepal (Mugula et al., 2001). The positive growth of lactic acid
bacteria in these types of foods shows that it is possible to use grain as a substrate for the growth of
probiotics. By obtaining all the benets from the fermentation process (improving the nutritional and
organoleptic characteristics of the food and reducing antinutritional factors), fruits and vegetables rep-
resent health-promoting foods thanks to the combination of probiotics and prebiotics naturally present
in their structure (Prado et al., 2008). Cereals, for example, may be an excellent substrate for the growth
of probiotics, and they can also be used as prebiotics for the presence of nondigestible carbohydrates
(Charalampopoulos et al., 2002).
Strains of Lactobacillus have been used in this regard for the production of a beverage made of single
and mixed fermented cereals. The microorganisms survived producing a large amount of lactic acid.
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193Probiotics and Prebiotics in Fruits and Vegetables
However, this large production of lactic acid and other organic acids shows that the enrichment of food
with probiotics can have a negative effect on its sensory characteristics. Therefore, in order to improve
both the survival of the microorganisms and also the sensory characteristics of the food, it is necessary
to conduct further studies (Rathore et al., 2012).
A yogurt-like beverage made of a mixture of cereals (rice, barley, emmer, and oat) and soy ours and
concentrated red grape must was produced adding two probiotic selected strains of L. plantarum (6E and
M6). Beverages processed with the mixture of rice and barley or emmer ours were preferred in terms
of textural, nutritional, and sensory properties (Coda et al., 2012). Ten panelists evaluated the product
and found that the drink had a very intense fruity smell, a dark color, and a very sour taste, with a grainy
texture (Coda et al., 2012).
Rößle et al. (2010) reported a study carried out on fresh-cut apple slice wedges enriched with a probi-
otic microorganism (LGG) and prepared by dipping the fruit in an edible buffer solution containing 1010
CFU/mL of LGG. The results showed that dipping apple wedges in a probiotic solution gives the product
good physicochemical and sensory characteristics of an acceptable quality and with an adequate number
of LGG adsorbed into the surface for a probiotic effect.
One aspect that must not be neglected in a food with probiotics is the survival of microorganisms. In
fact, food with probiotics must contain high doses of viable microorganisms throughout the shelf life of
the product.
Not all probiotic strains added to fruits and vegetables give good results in terms of survival. The
selection of strains of plant origin can help to overcome this and other technological challenges (Karasu
et al., 2010). In fact, numerous factors inuence microbial growth, such as salt, acidity, and pH. Some
strains of LAB, isolated from fermented vegetables and fruits, can be used as probiotics, because they
are able to resist high levels of acidity and NaCl during the storage period (Alzamora et al., 2005;
Betoret et al., 2003; Fleming and McFeeters, 1981; Renadheera et al., 2010). Microorganisms isolated
from fermented vegetables usually belong to the genera Lactobacillus, Leuconostoc, and Pediococcus.
Some of these have a resistance to acidic juices of the stomach and to bile and are comparable with
the probiotic strains isolated from humans and animals (Chiu et al., 2008; Fleming and McFeeters,
1981; Higashikawa et al., 2010). Potentially, probiotic microorganisms are isolated from many fermented
vegetables. Often, these microorganisms produce important compounds to ensure the microbiological
safety of food. Some microorganisms isolated from sauerkraut produce bacteriocins (nisin produced by
Lactococcus lactis) (Harris et al., 1992). Other microorganisms isolated from cucumbers (Atrih et al.,
1993) produce bacteria plantaricin C19, which is active against Listeria grayi, and those isolated from
olives (Delgrado et al., 2005; Mourad and Nour-Eddine, 2006) are producers of bacteriocins, which
are active against Weissella mesenteroides. Moreover, Lactobacillus strains that produce bacteriocins
active against E. coli, Staphylococcus aureus, and Bacillus cereus have been isolated from carrots (Joshi
et al., 2006). In vivo tests on mice have shown that strains of L. plantarum and Pediococcus pentosa-
ceus isolated from pickled cabbage have an inhibiting effect upon Salmonella. L. plantarum strains and
L. acidophilus have also proved to be good at adhering to the intestinal walls and showing resistance to
gastric juices and bile salts (Chiu et al., 2008; Wang et al., 2010).
These results led to the conclusion that the strains isolated from spontaneous plant fermentations can
be used as probiotics, having similar characteristics to those of the strains isolated from dairy prod-
ucts. Furthermore, the presence of proteins, minerals, ber, and other nutrients promotes the survival of
microorganisms, introducing a new concept of symbiotic food (Kalui et al., 2010).
Also soybean can be used as a carrier of probiotic microorganisms. Soybean-based foods (oil, milk,
and soy cheese) are rich in protein and can be used by vegetarians instead of meat, avoiding high cho-
lesterol levels in blood, preventing osteoporosis, and reducing the risk of contracting certain cancer
diseases. Moreover, these products have an unpleasant taste, are rich in antinutritional factors such as
phytates and enzyme inhibitors, and contain rafnose and stachyose in high concentrations that can
cause atulence. The fermentation process can enhance the avor, nutritional characteristics, and digest-
ibility as well as prolong the shelf life of the product (Rivera-Espinoza et al., 2010). In particular, pen-
tanal and n-hexanal are responsible for the undesirable beany avor of soymilk; a strain of B. breve
and some strains of L. acidophilus are able to metabolize these compounds in fermented soya products
(Champagne and Gardner, 2005).
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194 Benecial Microbes in Fermented and Functional Foods
Probiotic microorganisms (L. acidophilus MJLA 1, L. rhamnosus 100-C, L. paracasei ssp. paracasei
01, B. lactis BBDB2, B. lactis BB-12) have been also added into a nonfermented frozen vegetarian soy
dessert. The results highlighted that frozen soy dessert is a suitable probiotic food, with an excellent
viability of probiotic strains and acceptable sensory characteristics (Heenan et al., 2004).
A further study has also shown enhancement of the isoavones in soymilk supplemented with lactose
by using probiotic bacteria of the Lactobacillus and Bidobacterium genus during extended fermenta-
tion (Ding and Shah, 2010).
10.3.1 Probiotic Fermented Beverages
Fermented milk and drinks processed from fermented milk are among the most widely used vehicle
for probiotics, but also drinks based on fruit and vegetables have been tested as plausible vehicles for
probiotics, representing a new technological challenge. Fruit juices are potentially good vehicles of pro-
biotics. They are generally used by a large segment of the population, are appreciated and consumed by
individuals of all ages, and are often perceived as healthy (sometimes with added vitamins and minerals)
as well as refreshing beverages (Luckow and Delahunty, 2004; Suomalainen et al., 2006). The viability
of probiotic microorganisms in food matrices depends on several factors, such as storage temperature,
oxygen levels, pH, and the presence of competitor microorganisms, which must all be carefully evaluated
before they are added to foods (Gupta and Abu-Ghannaman, 2012). Moreover, it is important to consider
food matrix; L. acidophilus is reported to grow much better on soymilk than on cow’s milk (Champagne
and Gardner, 2005).
Also in fruit and vegetable juices, the tolerance to acidity is particularly important. These juices are
already naturally acidic; the fermentation process increases the acidity. Yoon et al. (2006) have reported
a study regarding the ability of L. plantarum, L. delbrueckii, and L. casei to survive in cabbage juice.
It was found that three strains of L. plantarum and L. delbrueckii are able to grow and maintain good
levels of vitality during several weeks of storage, resisting to the low pH, high acidity, and to refrigerated
storage at 4°C, with exception of L. casei, which loses its vitality after 2 weeks of storage.
In orange, pineapple, and cranberry juices, LGG, L. casei, B. lactis, Lactobacillus salivarius ssp.
salivarius, and L. casei and L. paracasei have shown high tolerance to pH, after being kept for 12
weeks at levels of 106 CFU/mL. All these strains are often used in the dairy industry for their tech-
nological strength. In this case, although all microorganisms have given good results, L. paraca-
sei NFBC 43338 is the best in terms of tolerance to acidity, resistance to pasteurization treatments,
resistance to pressure, and resistance to refrigerated storage (Sheehan et al., 2007). Also vegetable
juices have been tested to assess their suitability to be used as a carrier of probiotics. The carrot is
naturally rich in functional components (vitamins and minerals). Carrot juice seems to be suitable for
the growth of B. lactis and Bidobacterium bidum without the addition of other nutrients, remaining
viable at around 108 CFU/mL up to 24h. Carrot juice is rich in sucrose, glucose, and fructose, pos-
sible sources of carbon for bidobacteria. However, in this case, only glucose and sucrose have been
used by microorganisms. The content of fructose remained almost unchanged, indicating that for
bidobacteria this sugar does not constitute a source of nourishment. The slight change in carotenoid
content is, perhaps, due to the metabolic activity of microorganisms or to changes in pH, caused by
the production of lactic acid, and temperature occurring during the fermentation process. In any case,
the degradation of carotenoids was not excessive and the nutritional value of the product has not been
affected (Kun et al., 2008).
In cantaloupe melon juice, L. casei B-442 survived in high concentrations, due to the fact that this
juice contains high levels of fermentable sugars, like sucrose and fructose. In fact, the L. casei strain
can grow and develop without the addition of other sugars. Moreover, the microorganisms of the genus
Lactobacillus need amino acids and peptides derived from nucleic acids, vitamins, and fermentable
carbohydrates; therefore, even if the cantaloupe melon juice contains little protein, it is rich in amino
acids, such as aspartic acid, glutamic acid, arginine, and alanine that favor the growth and survival of
probiotics (Fonteles et al., 2012).
Tomato and tomato-based products can be considered as a healthy beverage, being a source of lyco-
pene, vitamins, and antioxidant. It has been reported that the probiotic strains of L. casei, L. acidophilus,
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195Probiotics and Prebiotics in Fruits and Vegetables
L. plantarum, and L. delbrueckii are resistant to low pH during storage at 4°C, surviving at levels of 106
108 CFU/mL in tomato juice. In tomato juice enriched with FOS, low pH did not inuence the lycopene
content, nor are its chemical properties modied. The addition of FOS also had a positive inuence on
the sensory characteristics of the product. In fact, in the juice enriched with FOS, the redness increased
signicantly; furthermore, after an analysis of sensory characteristics, it was observed that the avor had
improved while the off-avors decreased with the addition of FOS (Koh et al., 2010). Similar results were
also obtained for other juices. In pomegranate juice, L. plantarum and L. delbrueckii survived at their
maximum levels (2.9–3.9 × 108 CFU/mL) for 2 weeks of storage at 4°C with the number of microorgan-
isms decreasing after 4 weeks; L. paracasei and L. acidophilus lost their viability after the second week
in the same conditions (Mousavi et al., 2011).
A fermented beverage made from whole-grain oat was produced using L. plantarum B28. The prod-
uct, obtained after fermentation for 8h and stored at 4°C–6°C, was analyzed in order to determine
the beta-glucan content, naturally present in oat. Moreover, the microbial count has been performed to
determine the viability of the probiotic microorganism in the beverage, and nally, the effect of adding
different sweeteners on microbial growth was studied. During fermentation, the beta-glucan content
remained unchanged, indicating that the microbial culture did not ferment beta-glucan. The viable cell
counts at the end of the storage were 106–107 CFU/mL.
In order to sweeten the product, making it suitable for consumption by diabetics, several sweeteners,
such as sucrose, aspartame, sodium cyclamate, and saccharin, have been added to the probiotic drink. It
was found that the sweeteners did not affect the fermentation kinetics nor the viability of the probiotics.
The shelf life of this beverage was estimated at 21days of chilled storage (Angelov et al., 2006).
L. acidophilus was successfully added also to beetroot juice; in this case, the microorganism not only
maintained good vitality, but the juice obtained was also better in terms of the pigment, vitamin, and
mineral content, compared with the control without probiotic (Rakin et al., 2007).
In Africa and Asia, many fermented cereal-based foods have been produced by using lactic acid bac-
teria. It has been observed that the fermentation process improves the avor and aroma of the product
(Kalui et al., 2010).
In cashew apple juice, at the end of the cold storage period (42days), the viable count of probiotic
L. casei NRRL B-442 was higher than 8.00 log CFU/mL, without important loss of organism viability
throughout the storage period. Moreover, the juice was stable without any modication of the character-
istic yellow color. In addition, the activity of the enzyme characteristic of the fruit, and responsible for
browning, was well controlled without the addition of chemical compounds, such as sodium metabisul-
te (Pereira et al., 2011).
However, in some cases, the addition of probiotic microorganisms can negatively affect the sensory
characteristics of the food. Luckow et al. (2006) have reported the negative effect that the addition of
probiotic microorganisms (L. paracasei spp. paracasei NFBC 43338, LGG, L. casei DN-114001) had on
orange juice. The consumers have dened the juice with dairy, metallic, medicinal odor and with bitter,
acid, cooked avor. However, exposure to and familiarity with probiotic drinks, in addition to specic
information regarding the health benets of probiotic ingredients, helps improve consumer acceptance
and liking for the sensory characteristics of probiotic juices.
Another possible obstacle to the use of probiotics in fruit juices is an excessively low pH. Fruit and
vegetable juices are already acids; in addition, the fermentation process increases the acidity. A useful
method for raising the pH of the juice is with the addition of milk ingredients (Sheehan et al., 2007).
Furthermore, it is necessary to implement strategies that help to preserve the viability of probiotic
microorganisms, such as the addition of prebiotic components. Prebiotic ingredients extracted from fer-
mented cashew apple juice positively inuence the growth of L. mesenteroides and L. johnsonii. The
growth of these two microorganisms is better in apple juice with oligosaccharides than that observed in
juice containing only glucose and fructose as carbon sources (Vergara et al., 2010).
Therefore, several studies have shown that the use of probiotics in foods processed from fruits or veg-
etables is possible. However, the use of protective barriers and microencapsulation for preserving these
microorganisms is recommended.
Tables 10.1 and 10.2 summarize the main results regarding the technological and sensory effects of
probiotics and prebiotics in fruit- and vegetable-based foods.
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196 Benecial Microbes in Fermented and Functional Foods
TABLE 10.1
Technological and Sensory Aspects That Emerged from the Addition of Probiotics and Prebiotics in Fruit-Based Foods
Microorganisms Food Products Technological and Sensory Aspects References
L. paracasei ssp. paracasei NFBC
43338, LGG, L. casei DN-114 001
Orange juice The addition of probiotics in the juice had negative effects. The consumers have dened
the juice with dairy, metallic, medicinal odor and with bitter, acid, cooked avor.
Luckow et al. (2006)
L. johnsonii B-2178 Cashew apple juice The growth of the probiotic microorganism was threefold higher in apple juice with
oligosaccharides than that observed in the juice containing only glucose and fructose
as carbon sources.
Vergara et al. (2010)
LGG, L. acidophilus NCFM (free
cells and encapsulated)
Orange juice and fruit snack The samples with encapsulated organisms have shown a pH higher than samples free
from microorganisms. This aspect could be useful to reduce acidication and possible
negative sensory effects in orange juice and fruit snack.
Sohail et al. (2012)
L. casei DN 114001, LGG,
L. paracasei NFBC 43338, and
B. lactis Bb-12
Orange, pineapple, and
cranberry juices
Although all microorganisms gave good results, L. paracasei NFBC 43338 was the best
in terms of tolerance to acidity, resistance to pasteurization treatments, resistance to
pressure, and refrigerated storage.
Sheehan et al. (2007)
LGG Fresh-cut apple slices dipped
in an edible solution
containing a probiotic
Dipping apple wedges in probiotic solution imparts to the product good
physicochemical and sensory characteristics, with acceptable quality and with
adequate numbers of LGG adsorbed onto the surface for a probiotic effect.
Rößle et al. (2010)
L. casei NRRL B-442 Cantaloupe juice L casei B-442 survived in high concentrations. The reason for this is the high content of
fermentable sugar that contains the juice of cantaloupe like sucrose and fructose.
Fonteles et al. (2012)
An enhancement of the isoavones in soymilk supplemented with lactose during
extended fermentation has been observed.
L. casei NRRL B-442 Cashew juice During the 42-day storage, any loss of organism viability was not observed and the
juice was stable without any modication of the characteristic yellow color.
Pereira et al. (2011)
L. casei, L. paracasei,
L. plantarum, L. acidophilus,
L. delbrueckii
Pomegranate juice L. plantarum and L. delbrueckii were capable to survive for 2 weeks of storage at 4°C
at their maximum levels (2.9–3.9 × 108 CFU/mL), decreasing the number of
microorganisms after 4 weeks; L. paracasei and L. acidophilus failed their viability
after the second week in the same conditions.
Mousavi et al. (2011)
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197Probiotics and Prebiotics in Fruits and Vegetables
TABLE 10.2
Technological and Sensory Aspects That Emerged from the Addition of Probiotics and Prebiotics in Vegetable-Based Foods
Microorganisms Food Products Technological and Sensory Aspects References
L. plantarum 6E and M6 Yogurt-like beverage made of
a mixture of cereals (rice,
barley, emmer, and oat) and
soy ours and concentrated
red grape must
Beverages processed with the mixture of rice and barley or emmer ours were
preferred in terms of textural, nutritional, and sensory properties.
Coda et al. (2012)
L. plantarum B28 Fermented beverage based
on oat
L. plantarum gave good results in terms of survival of microorganisms and sensory
characteristics during all the period of storage.
Angelov et al. (2006)
Bidobacterium strains: B. lactis,
B. bidum
Pure pasteurized carrot juice The microorganisms remained viable at around 108 CFU/mL up to 24h. The addition
of probiotic leads to a production of lactic acid but without excessive degradation of
carotenoids, not affecting the nutritional value of the product.
Kun et al. (2008)
L. casei A4, L. delbrueckii D7, and
L. plantarum C3
Cabbage juice L. plantarum and L. delbrueckii were able to grow and maintain good levels of vitality
during several weeks of storage, resisting to the low pH, high acidity, and refrigerated
storage at 4°C. L. casei, however, has proved weaker and therefore has lost its vitality
after just 2 weeks of storage.
Yoon et al. (2006)
L. acidophilus MJLA 1, L. rhamnosus
100-C, L. paracasei ssp. paracasei
01, B. lactis BBDB2, B. lactis BB-12
Nonfermented frozen soy
dessert
Frozen soy dessert is a suitable probiotic food, with an excellent viability of probiotic
strains and acceptable sensory characteristics.
Heenan et al. (2004)
L. plantarum NCIMB 8826 and
L. acidophilus NCIMB 8821
Malt beverage The considerable production of lactic acid and other organic acids has highlighted the
possibility of a negative sensory effects in product enriched with probiotics.
Rathore et al. (2012)
L. casei, L. acidophilus,
L. plantarum, L. delbrueckii
Tomato juice In tomato juice enriched with FOS, low pH did not inuence the lycopene content, nor
are its chemical properties modied. The addition of FOS also had a positive
inuence on the sensory characteristics of the product. In fact, in the juice enriched
with FOS, the redness increased signicantly; furthermore, it was observed that the
avor had improved while the off-avors decreased with the addition of FOS.
Koh et al. (2010)
Lactobacillus and Bidobacterium
strains
Soymilk An enhancement of the isoavones in soymilk supplemented with lactose during
extended fermentation has been observed.
Ding and Shah (2010)
L. acidophilus Beetroot juice The microorganism maintained a good vitality. The product with probiotic was found
better in terms of pigments, vitamins, and minerals content with respect to the control.
Rakin et al. (2007)
LAB Fermented cereal-based food The fermentation process improves the avor and aroma of the product. Kalui et al. (2010)
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198 Benecial Microbes in Fermented and Functional Foods
10.4 Microincapsulation
Stability, survival, and resistance to gastric juices and bile salts are essential for probiotic microorgan-
isms, since these qualities can inuence their benecial properties. The therapeutic value of probiotic
bacteria generally depends on the viability of these microorganisms. However, the industrial pro-
cesses used for food preparation, such as freezing, storage temperatures, values of acidity, and oxygen
content of food matrices, are not always optimal to ensure the functionality of probiotics (Heidebach
et al., 2012).
In order to enhance probiotic survival, microencapsulation successfully creates physical protection
during food storage and favors the passage of probiotic through the digestive tract. Microencapsulation
is dened as “the technology of packaging of solid, liquid or gaseous materials miniaturized in capsules
that can release their contents in a controlled manner and only under certain conditions” (Burgain et al.,
2011). Two types of capsules are available:
1. The reservoir type, in which a shell covers the encapsulated substance
2. The matrix type, in which the active agent is dispersed within and on the surface of a support
material
A third type of capsule derives from the combination of these two types, in which the active material is
coated but also is dispersed within and on the surface of a matrix (Burgain et al., 2011).
The capsule containing the microorganisms is composed of a substance that forms a thin, yet resistant,
semipermeable membrane, to protect the content during the passage into the stomach.
Different materials can be used for various microencapsulation techniques: polysaccharides derived
from seaweed (carrageenan and alginate), plants (starch and gum arabic), substances of bacterial origin
(gellan and xanthan gum), and animal protein (casein). The microencapsulation methods are described
in the following.
Extrusion method. This is a method that allows to perform the encapsulation causing minimal damage
to the microbial cells. The rst step consists of adding probiotics to a hydrocolloid solution, and then the
solution is dripped through a syringe needle or nozzle. The dimensions of the nozzle affect the diam-
eter of the microcapsules. Encapsulation materials that are usually used for this technique are alginate,
k-carrageenan and k-carrageenan plus locust bean gum, xanthan plus gellan, alginate plus corn starch,
and whey proteins (Huq et al., 2013).
Emulsion method. Unlike the extrusion technique, in this case, the diameter of the beads can be
controlled and is lower (25µm–2mm). This method requires the use of a vegetable oil (soy oil, sun
ower oil, corn oil, or millet or light parafn oil) for emulsion formation. The oil is mixed with a
small amount of cell/polymer slurry and is agitated to form an emulsion. The polymer becomes
insoluble in solution with the addition of calcium chloride and forms gel particles in the oil phase.
The use of emulsiers that guarantee the formation of beads with a smaller diameter is also possible
(Huq et al., 2013).
Drying method. The drying of the encapsulated mixture can be performed by various methods:
freeze-drying, spray drying, and uidized bed drying. Generally, the rate of survival of microorgan-
isms with these techniques is in the range of 70%–85%. However, it is complicated to maintain long
product stability. The technique of freeze-drying due to minimal cellular injury but it’s necessary to use
a cryoprotective agent to protect cells from cold damage. Spray drying allows to treat large volumes of
solutions; the technique is relatively cheap, but the loss of viability of the cells is considerable due to the
phenomena of heating and dehydration. Best results were obtained by changing the spray-drying tech-
nique. The method consists of milk fat droplets containing powder coating particles of freeze-dried cells
with polymers of whey proteins, in a condition where an emulsier is used (Huq et al., 2013). Picot and
Lacroix (2004) have reported that a valid method, on the industrial scale with respect to cell viability and
economics, is dispersing cells of Bidobacterium spp. in a suspension of whey protein containing milk
fat droplets and then proceeding with the spray drying.
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199Probiotics and Prebiotics in Fruits and Vegetables
Irrespective of the technique used for encapsulation (extrusion, emulsion, or spray drying), microen-
capsulation is one of the most effective techniques for the defense of probiotic viability.
The success and the validity of the microencapsulation technique is documented by a series of studies
showing that there is genuine preservation of the vitality of microorganisms compared to the products
in which the probiotics are free (Kebary et al., 1998; Rokka and Rantamaki, 2010; Saxelin et al., 2010;
Shah and Ravula, 2000). Microentrapment in gel beads has shown to be very effective to maintain the
probiotic viability of frozen foods (Champagne and Gardner, 2005).
Even microencapsulation technology applied to fruits or vegetables has produced good results.
Microencapsulation, with alginates and chitosan, protects microorganisms from inhibitors, such as acids
and avonoids (Koo et al., 2001), allowing high levels of vitality and good efciency during fermentation
of the encapsulated bacteria. K-carrageenan and Ca alginate were used successfully for the immobiliza-
tion of L. acidophilus in banana puree (Tsen et al., 2003) and in tomato juice (King et al., 2007; Tsen
et al., 2008). In particular, in the study reported by King et al. (2007), sodium alginate and calcium
chloride were used to form the immobilizing matrix for the entrapment of cells of L. acidophilus. The
results showed that the viability of encapsulated microorganisms is higher in tomato juice compared
with free cells. Moreover, the juice containing the encapsulated cells was also more pleasant than the
juice containing the free cells. This is probably due to the higher viable cell number of immobilized
cells and because unfavorable deterioration reaction were inhibited during storage. Excellent results in
terms of survival and sensory characteristics of the product formulated with microencapsulated probiotic
L. acidophilus have been also reported by Tsen et al. (2008) for tomato juice.
The size of the capsules (generally ranging from 1 to 5 μm diameter) is particularly signicant. The
diameter of the capsules should be wide enough to ensure the protection of the bacterial content but, at
the same time, sufciently small not to create negative changes of the sensory characteristics of the food.
Indeed, sensory studies have shown that high concentrations of spherical particles in the food product
give sensations of roughness and graininess, which are disfavored by the consumer (Engelen et al., 2005;
Imai et al., 1995).
The encapsulated organisms are often added to yogurt and cheese, with a consequent evident increase
in the viability of the microorganisms during the storage period. Nevertheless, the protective effect is
only obtained with capsules of 0.2–3mm. With capsules of this size, the negative impact on the sensory
characteristics of the product is guaranteed.
A new technique for the encapsulation uses a separate impinging aerosol of sodium alginate solution
and calcium chloride cross-linking solution to produce water-insoluble cross-linked alginate micro-
beads with an average diameter less than 40 μm (Sohail et al., 2012). This technique does not require
the use of heat or solvents; therefore, it can also be used for heat- or solvent-sensitive materials. This
technique has been used for encapsulation of LGG and L. acidophilus NCFM in orange juice, with a
pH of 3.80, and in pear- and peach-based fruit snacks. From this study, the following important aspects
have emerged:
Microencapsulation protects the viability of LGG; however, also, free cells have shown a pro-
pensity for survival. Conversely, this technique of encapsulation did not favor L. acidophilus
NCFM survival. Therefore, it is clear that the ability of microorganisms to survive in food
matrix largely depends on the characteristics of the strain; the microorganism L. rhamnosus is
suitable for growth in acidic environments even without protective techniques of microencap-
sulation, while L. acidophilus is decidedly more sensitive.
Another aspect that emerged regards the effect of microencapsulation on the variation of
pH in the food matrix. In a comparison of the pH values of orange juice and fruit-based
products produced with free and encapsulated probiotic microorganisms (LGG, L. acidophi-
lus NCFM) throughout a 9-day storage period, it was found that the encapsulated micro-
organisms did not induce as much lowering of pH as free microorganisms present in the
food matrix (Sohail et al., 2012). Therefore, microencapsulation in alginate beads could be
effective in reducing acidication and could improve the sensory properties of fruit-based
products.
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200 Benecial Microbes in Fermented and Functional Foods
Even if microencapsulation may be a good technique for protecting the viability of microorganisms,
there are still many obstacles to overcome to improve encapsulation techniques:
Protection from stress acids must be ensured.
The capsules should be digested in the intestine so that the content can be released and our
health can benet.
The capsules must have optimal dimensions in order to serve as valid barriers, but at the same
time they should not disturb the palate of the consumer.
The technical choices for the encapsulation must not reduce the number of live cells or cause
sublethal damage in humans.
At the moment, no encapsulation technique can fulll all these requirements (Heidebach et al., 2012).
10.5 Challenges for Probiotic Foods Formulation
Although research has made great strides, there are many challenges that must be addressed when decid-
ing to make a food that contains probiotics. For example, a consumer who buys a food with probiotics,
rightly, expects that food maintains its characteristics unchanged throughout the shelf life; this includes
that the microorganisms must remain viable during this period. Different aspects have to be considered:
the characteristics of the food matrix, the effects of storage conditions of foods on the microorganisms,
and the choice of microbial strains to be used in different foodstuffs. The choice of the probiotic strain
is a crucial step. The pH value seems to be a critical factor in the stability of probiotic strains during
storage; L . acidophilus cultures are generally more resistant to acid environments than bidobacteria.
Moreover, it is also necessary to take into account the fact that a microorganism capable of withstanding
the acid pH of a food may not be able to resist the acid pH of the stomach. In fact, the two aspects are not
always correlated. For example, some microorganisms, such as L. lactis, S. thermophilus, and L. mes-
enteroides, grow well in foods with acidic pH, but are not able to grow in the gastrointestinal tract, thus
losing their function of probiotics. Also, the time of exposure to low pH is an important aspect. A strain
able to resist to the acid pH of the stomach may not be able to resist the acid pH of the product during the
storage period. At the moment, it is not well known if there is a link between the ability to survive short-
time exposures to high-acid environment and the ability to survive long term in fermented products. In
this regard, Chavárri et al. (2010) reported a study on microencapsulation of probiotic microorganisms
in simulated gastrointestinal conditions. In this study, chitosan was used as a coating material to improve
encapsulation of Lactobacillus gasseri and B. bidum in calcium alginate beads. The encapsulation in
this type of microspheres signicantly increased the viability of the microorganisms. In particular, it
was found that the microorganisms encapsulated in chitosan-coated alginate beads are able to withstand
the conditions of the gastrointestinal tract, maintaining throughout the exposure time (2h) a viability of
95%. The present study showed that chitosan is a great ingredient that increases the stability of alginate
microcapsules in adverse conditions. The presence of chitosan reduces the porosity of the capsules of
alginate and decreases the leakage of the encapsulated probiotic, and accordingly probiotics are more
stable during the passage through the gastrointestinal tract.
Another aspect to be considered is the effect that other microorganisms present in the medium may
have on probiotics. Probiotics, for example, are particularly sensitive to the presence of oxygen. Probably,
the toxic effect of oxygen is due to the accumulation of intracellular hydrogen peroxide favored by the
presence of oxygen. Some microorganisms are capable of producing hydrogen peroxide and drop it in
the middle, so the presence in the microorganism with this attitude may adversely affect the survival
of the probiotic. In this case, it could be useful to replace the microorganism that produces hydrogen
peroxide with one that does not. In fermented milks, the elimination of L. delbrueckii ssp. bulgaricus
has had positive effects on the survival of L. acidophilus. Another possibility would be to add to the
food antioxidants such as ascorbic acid. From these considerations, it is clear that other studies to assess
what is the best combination of microorganisms to be used for the production of probiotic foods are
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201Probiotics and Prebiotics in Fruits and Vegetables
still needed (Champagne and Gardner, 2005). Also, the presence of starter cultures in foods used for
technological purposes (acidication, avor, texture) can represent an obstacle for the growth of pro-
biotic strains; the common strategies to reduce competing lactic cultures and to improve the growth
or survival of probiotic cultures are the omission of a portion of the starter strains and changes in the
respective inoculation rates; in fermented soya milk inoculated with lactic acid starter and bidobacte-
ria probiotics, this problem has been overcome by the addition of cysteine in the product (Champagne
and Gardner, 2005).
Also, food processes that include a heating step above 65°C are highly detrimental for probiotic viabil-
ity; therefore, it is necessary to add the cultures after thermal treatment. Moreover, microencapsulation
of the dried probiotic cultures with lipids could represent a way of protecting the cells against heat treat-
ment (Champagne and Gardner, 2005).
Another important aspect to consider is the dose of microorganisms to be added to the food in order
to guarantee the colonization of the gastrointestinal tract. The minimum recommended level of probiotic
in foods is 106 CFU/g of food product or 107 CFU/g at point of delivery or be eaten in adequate amounts
to yield a daily intake of 108 CFU/g (Chávarri et al., 2010). What still needs to be considered is the effect
of the composition of the food on the viability of probiotics. It is difcult to determine the exact number
of microorganisms; at the moment, the best approach is still to be based on scientic studies conducted
on specic foods (Champagne and Gardner, 2005).
10.6 Conclusions
The market of functional foods is growing; this growth is fueled by an increased level of consumer atten-
tion to diet. The consumption of fruits and vegetables is generally associated with the idea of well-being,
as these foodstuffs are rich in minerals and vitamins, antioxidants, and ber; moreover, the addition of
probiotic microorganisms enhances the quality of these foodstuffs. In some cases, fruits and vegetables
also represent a natural source of prebiotics, which have a protective function toward probiotic microor-
ganisms, preserving their vitality during shelf life of the product. Therefore, fruits and vegetables create
a new concept of symbiotic food. Several studies have shown that foods made with fruits and vegetables
can be successfully used as carriers of probiotics, maintaining good levels of viability of probiotic micro-
organisms and good sensory characteristics. Microencapsulation of microorganisms can contribute to
the maintenance of the functionality of food. However, as shown in this chapter, not all organisms show
the same behavior under certain conditions. Therefore, in addition to using protective techniques such as
microencapsulation, it is necessary to have a thorough understanding of the food and the physiological
characteristics of the probiotic strain. There are still many challenges ahead, and in any case, the choice
of probiotic strain to be used in food is essential.
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This study evaluated the viability of Lactobacillus plantarum LP299v or Lactobacillus rhamnosus GG in vegetable appetizer, as well as the resistance of the strains to the gastrointestinal tract (GIT) simulated in vitro. Control appetizer and added of probiotic strains were prepared and remained at 8 °C for 90 days. There was no difference in the L* and b* between the treatments and throughout the storage time. The control appetizer presented higher pH and lower acidity compared to probiotic appetizer. Vegetable appetizer showed counts of L. plantarum or L. rhamnosus higher than 7.42 Log CFU/g and 8.84 Log CFU/g respectively, along the refrigerated storage, being verified greater viability for L. rhamnosus (p < 0.05) with no reduction in the counts of both microorganisms over time (p > 0.05). Mean scores above 6.0 (“slightly appreciated”) were attributed to sensory analysis. The appetizer containing L. rhamnosus had a higher preference among consumers. In the in vitro GIT test, considering the consumption of a 100 g portion, in the time 90 days, approximately 8.67 Log CFU/g and 9.53 Log CFU/g of L. plantarum or of L. rhamnosus respectively, would be available to promote consumer benefits, which makes the appetizer apt to be considered probiotics.
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The production of value-added and/or functional juices has increased significantly in recent years, following an increased consumer demand to promote health and/or prevent disease through diet and nutrition. Micro and nano-encapsulation are promising technologies to protect and deliver sensitive compounds, allowing a controlled release in the target sites. This paper offers an overview of current applications, limits and challenges of encapsulation technologies in the production of fruit and vegetable juices, with a particular emphasis on products derived from different botanical sources.
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Ice-cream containing probiotic bacteria was produced by mixing fortified milk fermented with probiotic strains with an ice-cream mix, followed by freezing. Four different strains of probiotic bacteria were used. Each strain was grown (37°C, 12 h) in UHT semi-skimmed milk fortified by the addition of 1% glucose and 1% tryptone. The fermented milk was added as a 10% addition to an ice-cream mix. The complete mix was frozen in a soft- ice freezer and hardened at ÷20 °C. Ice-cream mixes with and without the addition of glycerol were produced to ascertain whether this had a protective effect against freezing of the probiotic bacteria. Viable counts of probiotic bacteria were made immediately after mixing and after freezing, as well as after 1, 4, 16 and 52 weeks of storage at -20 °C. The ice cream samples were organoleptically assessed for probiotic flavour, firmness, chewiness. sourness, off-flavour, iciness and total impression. During freezing, or shortly afterwards, the viable count declined by 0.7-0.8 log cfu/g. The viable count did not change significantly during 52 weeks of frozen storage (p<0.05) and remained above the recommended minimum limit of 106 cfu/g. The incorporation of glycerol in the ice-cream did not improve the survival of the strains. All the ice-cream samples received a high score in the organoleptic evaluation; the probiotic taste was not found to be particularly noticeable. Lb. reuteri containing ice-cream was significant more sour and attained a higher "probiotic flavour" than the other ice-cream samples.
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Purpose. Ca-alginate entrapped Lactobacillus acidophilus was used to ferment tomato juice and enhance the survival of the bacteria in the product. Methods. L. acidophilus was immobilized in gel beads with diameters of about 26 mm. Tomato juice was made from fresh raw tomatoes, followed by fermentation for 80 h with free and immobilized cells. Results. Immobilized cells leaked from the gel beads and proliferated in the juice during the fermentation; the final viable cell number reached 10 7 CFU/mL in the juice and above 10 10 CFU/mL-gel in gel beads. Free cells reached about IO 9 CFU/mL during fermentation. Immobilized cells endured the adverse conditions in tomato juice; furthermore, viable cell numbers and sensory score results were higher compared with free cells. The viable cell counts of immobilized L. acidophilus were maintained at 10 7 CFU/mL-gel in the fermented tomato juice after 10 weeks of cold storage at 4°C, compared with 10 4 CFU/mL of free cells. Conclusions. Ca-alginate immobilized L. acidophilus enhanced the viable cell number and improved the sensory quality of fermented tomato juice. Our findings could be applied to the development of probiotic tomato juice.