Probiotics in Functional Foods: © 2019 Elsevier Inc. All Rights Reserved
Probiotics in Functional Foods: © 2019 Elsevier Inc. All Rights Reserved
Probiotics in Functional Foods: © 2019 Elsevier Inc. All Rights Reserved
Sandrayee Brahmaa, Muhammad Bilal Sadiqb, and Imran Ahmadc, a Department of Food Science & Technology, University of
Nebraska-Lincoln, Lincoln, NE, United States; b School of Life Sciences, Forman Christian College A Chartered University, Lahore,
Pakistan; and c Food Agriculture and Biotechnology Innovation Lab (FABIL), Florida International University, Biscayne Bay Campus,
North Miami, FL, United States
© 2019 Elsevier Inc. All rights reserved.
Introduction 1
Current Market Status and Trends 2
Pro-biotics and Pre-bioticsdSynbiotic Phenomenon 3
Foods Containing Probiotics 3
Dairy Products 4
Meat Products 6
Cereal-Based Products 6
Fruit and Vegetable-Based Products 7
Food Waste Materials 7
Recent Developments in the Isolation, Utilization, and Production of Probiotic Strains 8
Delivery of ProbioticsdChallenges and Opportunities 8
Probiotic Survival During Processing and Storage 11
Conclusions and Future Directions 13
References 13
Further Reading 16
Introduction
Nutraceuticals and functional foods have gained considerable attention across the world because of their potential health benefits.
Consumers are becoming increasingly aware that an inadequate dietary plan could lead to malnutrition and disease (Kumar et al.,
2017). More than 20 years ago, Stephen DeFelice, the founder of the Foundation of Innovation Medicine, New Jersey, USA, first
coined the term “nutraceutical” which is a hybrid of the words ‘nutrient’ and ‘pharmaceutical’(DeFelice, 1995). The term may be
defined in general as naturally occurring foods, or parts of the food having physiological benefits or protecting human health against
chronic diseases (DeFelice, 1995; Mattila-Sandholm et al., 1999). Now the term “nutraceutical” encompasses a broad range of prod-
ucts such as functional foods, fortified foods, dietary supplements, isolated and purified nutrients, herbal products and specific diets
which are sold in medicinal forms and may not be usually related with food (HealthCanada, 1998; Nworu et al., 2014; Jacobs and
Tapsell, 2013; Shahidi, 2012; Venhuis et al., 2016). The term may also include processed foods and beverages (Udenigwe and
Aluko, 2012). Although the definitions may vary between different countries or different health-related professional organizations,
the proposed meanings are mostly covered by all the associated definitions (Wildman et al., 2016). According to the European
Nutraceutical Association (ENA), nutraceuticals are defined as “synthetic substances or chemical compounds formulated for specific
indications,” and are considered opposite to pharmaceuticals.
Similarly, the term “functional foods” has contradictory definitions. Although both the terms “nutraceutical” and “functional
foods” can be used interchangeably, there are significant differences in the meaning between these two. Confusion may arise
when the “functional food” for one consumer serves as “nutraceutical” but does not serve the same purpose for another (Kalra,
2003). Definitions of the term “functional food” were established well before the term “nutraceutical” was defined. This term first
appeared in the Japanese literature in 1984, to differentiate the primary function of nutrition from the secondary function of pref-
erence. Health Canada has defined functional food as “is similar in appearance to, or maybe, a conventional food that is consumed
as part of a usual diet, and is demonstrated to have physiological benefits and/or reduce the risk of chronic diseases beyond basic
nutritional functions, i.e., they contain bioactive compounds” (HealthCanada, 1998). According to the International Life Sciences
Institute of North America (ILSI, 2006), functional foods can be described as “foods that by physiologically active food components
provide health benefits beyond basic nutrition” (ILSI, 2006). Whether it is to be called a nutraceutical or functional food, it is the
bioactive component possessing physiological benefits that can be further used either as functional food ingredients or to produce
nutraceuticals (Kumar et al., 2017).
Understanding the safety and efficiency of nutraceuticals and functional foods is critical, and nowadays this requires novel
“omics technological tools” such as nutrigenomics, nutrigenetics, and proteomics to unravel potential insights into the series of
biochemical changes and complex interactions that accompany dietary interventions with these nutraceuticals or functional foods
(Bagchi et al., 2015). Metabolomics is also an approach that can be used to identify genes and gene-related functions as well as
health-promoting compounds and metabolites for developing nutraceuticals and functional foods (Kumaraswamy et al., 2011;
Pushpa et al., 2014). Currently, one line of research into nutraceuticals and functional foods has progressed toward the concept
Enhanced Enhance
Cholesterol Vitamin B
Detoxification nutrient lactose
reduction production
bioavailability tolerance
Immunomodulation
Reduce intestinal Functional values of probiotic
and
pathogen load enriched foods
immunostimulation
Enhance
Anti- Reduce
absorption of Treatment of Regulation of
inflammatory incidence of
Fe, Ca and diarrhoea intestinal flow
effect colon cancer
Mg
of dietary supplementation with probiotics and prebiotics and their influence on the composition of the gut microbiota (Krumbeck
et al., 2016). Therefore, it is critical to have a thorough understanding of the biological nature and health claims of these probiotic
products both for consumers and health professionals. An overview of common functionalities and health outcomes of probiotic-
enriched foods is depicted in Fig. 1.
This review focusses on the current state-of-play and the latest literature on probiotics, including current and future market
trends, currently available natural probiotic products, novel strategies for isolation and stabilization of probiotics, and remaining
challenges. In particular, the review explores the production and developmental issues of probiotics with a focus on strategies for
isolation of probiotics, byproduct utilization, and the impact of processing on probiotics incorporated in food products. The review
is illustrated with case studies that highlight and compare different innovation strategies for developing food products using
different probiotic cultures.
The USA is the world’s largest market for health foods, with an estimated value of USD253 billion in 2018, followed by Asia and
Europe. However, for the rest of the world, when the nutraceuticals category includes functional foods and dietary supplements,
driven by a trend toward natural ingredients, the market has a projected value of about USD45.6 billion in 2022. Probiotics are
Probiotics in Functional Foods 3
viewed as the fastest-growing specialty nutritional ingredient with net sales of USD2.4 billion in 2018, while omega-3 products are
expected to grow from USD1.2 billion to USD1.4 billion over the next several years.
Additionally, structured lipids, amino acids, peptides and proteins, phytochemicals and plant extracts, minerals, vitamins as well
as fibers and specialty carbohydrates are among other active ingredients (Augustin and Sanguansri, 2015). These products mostly
target populations such as infants, young children, teens, and older adults (Augustin and Sanguansri, 2015). For the general pop-
ulation, aged 18–48, “health-enhancing” foods have been consistently driving sales over the past five years in the functional food
and beverage sector.
The health potential of probiotics has a long history dating back more than 100 years. It was Metchnikoff (1910) who first reported
his observations on a group of individuals in Bulgaria who ingested fermented milk products (containing probiotic strains, albeit
not known at the time) on a daily basis, and he associated this consumption with their longevity. It wasn’t until recently that the
term “probiotic” was first published in Science, as “growth-promoting factors produced by microorganisms” (Lilly and Stillwell,
1965). The term has evolved and in the mid-1970s was defined as “organisms and substances which contribute to intestinal micro-
bial balance” (Parker, 1974). Later in 2012, the FAO/WHO working group established the definition of “probiotics” as “live micro-
organisms that, when administered in adequate amounts, confer a health benefit on the host,” which further confirms that the
concept of probiotic is a strain-specific characteristic. This latter definition is widely accepted by organizations such as the Interna-
tional Scientific Association for Probiotics and Prebiotics (ISAPP), Codex, the Institute of Food Technologists (IFT), the World
Gastroenterology Organization (WGO) and the European Food Safety Authority (EFSA) (Hill et al., 2014).
Prebiotics is a term first defined some 20 years ago as “functional non-digestible food ingredients” which selectively affect the
host by stimulating the growth and activity of one or a limited number of bacteria/probiotics in the colon, thereby improving host
colon health (Gibson and Roberfroid, 1995). These prebiotic substrates, when metabolized by the gut microbiota in the colon, yield
beneficial short-chain fatty acids, which in turn lower the pH, and trigger mucin production by colonocytes, and promote the
production of immunomodulatory cytokines (Preidis and Versalovic, 2009).
The concept of synbiotics was first envisioned by Gibson and Roberfroid (1995) about twenty years ago. A symbiotic is essen-
tially a combination of probiotics and prebiotics that act in synergy leading to the synbiotic being more active than the individual
constituent prebiotics or probiotics alone.
Complementary synbiotics are those which contain probiotics and prebiotics and each is responsible for a specific health effect,
and such effects are mostly additive (Kolida and Gibson, 2011). When a prebiotic and a probiotic are introduced together, any
synergistic synbiotic phenomenon can lead to an unpredictable outcome and will depend on an individual’s gut microbiota
composition. This phenomenon is referred to as “responders/non-responders,” representing how an individual’s gut microbiota
respond to prebiotic dietary interventions (Brahma & Rose (2019); Davis et al., 2011; Korpela et al., 2014; Kovatcheva-Datchary
et al., 2015; Salonen et al., 2014).
Brahma & Rose (2019) outlined the “responder/non-responder” phenomenon and its relation to human health. A considerable
difficulty is encountered to establish beneficial health impact of a dietary treatment due to this phenomenon because the response
to the prebiotic treatment not only depends on the taxonomic or functional composition of the microbiota, but also on the host’s
factors, such as digestive enzyme, food transit time, environmental constraints restricting the growth of certain bacterial species. The
synergistic synbiotics provide a prebiotic substrate that supports the growth of a specific probiotic strain not initially present in the
gastrointestinal tract (Kolida and Gibson, 2011). This approach of introducing a prebiotic substrate addresses the problem of
“responders/non-responders” assuming the target strain reaches the colon. One of the critical limitations of this approach is the
low ecological success of the probiotic strain (Krumbeck et al., 2015). Despite limited research on synergistic synbiotics, a significant
number of studies have focused on synbiotic applications only (Krumbeck et al., 2016; Shukla et al., 2011; Tomar et al., 2015). The
most common probiotic components cited in these studies are lactobacilli and bifidobacteria, inulin, dietary fibers and various
other oligosaccharides as the prebiotic components (Krumbeck et al., 2016; Shukla et al., 2011). The commonly used probiotics
available in the literature are L. johnsonii, Lactobacillus acidophilus, B. longum, Bifidobacterium animalis, Bifidobacterium infantum,
L. brevis and L. fermentum, while the common prebiotics may include inulin, galactooligosaccharides (GOS), xylooligosaccharides,
lactulose, lactitol, and fructooligosaccharides (FOS) (Tomar et al., 2015). Examples of the commonly available synbiotic combina-
tions include Bifidobacteria þ GOS, Bifidobacteria þ FOS, Lactobacilli þ lactitol, and Lactobacilli þ FOS or inulin (Tomar et al.,
2015).
Despite abundant health claims made for synbiotic applications in the literature, it is surprising that regulatory agencies have
approved none of these claims across the US and Europe (Krumbeck et al., 2016). However, more research needs to be conducted
into the mechanics of synbiotics to establish their therapeutic health benefits for personalized treatments.
Humans have used probiotics, albeit unknowingly, since the origin of fermented milk and fermented foods thousands of years ago.
A deeper scientific understanding of probiotic-mediated health benefits has only been explored relatively recently, and initially
4 Probiotics in Functional Foods
-Acidophilus
milk
-Acido-whey Fermented Non-fermented
-Ice-cream products products
-Lassi
-Cheese
-Curd
-Frozen
synbiotic
dessert Cereals Fruits Vegetables Meat and Fish Soy
-Yogurt, etc.
inspired by the work of Metchnikoff in the early part of the 20th century. He reported that imbalance in the gut had adverse effects
on health and he further suggested that consumption of fermented milk could help treat this condition (Kumar et al., 2015).
Fermentation has been used for ages as a preservative technique for various foods. Previously, fermented dairy products were devel-
oped only due to the spontaneous activity of indigenous microorganisms in milk, but modern fermentation techniques involve the
addition of specific starter culture with defined characteristics to ensure the quality of the fermented product. The selection of
a particular probiotic as a starter culture is based on its influence on organoleptic characteristics and long-term food product safety
(Hill et al., 2017). Among non-dairy probiotic foods, fermented fruits, vegetables, cereals, and soy-based products are gaining popu-
larity (Gawkowski and Chikindas, 2012; Martins et al., 2013). An overview of probiotic food products is given in Figs. 2 and 3
Dairy Products
Dairy products are the most widely used source of probiotics (Granato et al., 2010). The isolation of novel lactic acid bacteria (LAB)
from ethnic fermented milk products has always been a valuable approach to obtaining indigenous strains of probiotics (Hill et al.,
2017). Ethnic fermented milk products, such as matsoni of Armenian origin and kule naoto of Maasai in Kenya, contained undoc-
umented microorganisms that were later isolated and identified by genetic sequence-based analysis (Bokulich et al., 2015; Ogun-
toyinbo et al., 2016). Ethnic sources of probiotics can be of great value due to their availability, wide acceptance and being
economical. Shubat, for example, a camel milk fermented product of Kazakh origin, was found positive for hypoglycemic activity
in type II diabetic rats (Manaer et al., 2015).
Similarly, Raabadidan Indian fermented milk beverage has been reported as a rich source of lactobacilli with a hypocholestero-
lemia effect (Yadav et al., 2016). Kefir is yet another fermented milk product with a slightly acidic taste that originated in Eastern
Europe (Serafini et al., 2014). The freeze-dried kefir starter culture, traditional kefir grains and residual of kefir grains can be used to
Probiotics in Functional Foods 5
Figure 3 Categories of food for delivery of encapsulated probiotics (De Prisco and Mauriello, 2016).
ferment milk to produce commercial kefir (Bensmira et al., 2010). Kefir grains are used as a starter culture for milk fermentation and
are composed of a mixture of lactic acid bacteria (LAB), yeast and acetic acid bacteria (Chen et al., 2015).
Lyophilized kefir cultures provide high viability and fermentation efficiency, indicating their potential in the dairy industry (Ben-
smira et al., 2010). Kefiran is a major heteropolysaccharide, composed of glucose and galactose, present in kefir grains and produced
primarily through the action of Lactobacillus kefiranofaciens (Zajsek et al., 2011). Kefiran has been reported to improve rheological
properties of acid milk gels (Zajsek et al., 2013), and is associated with various biological activities including antitumor, antifungal,
antibacterial, immunomodulation, antioxidant and anti-inflammatory effects (Chen et al., 2015; Serafini et al., 2014; Wang et al.,
2008).
Several studies have reported clinical evidence about health-promoting effects of probiotics; claiming probiotic intake in the diet
can prevent or treat various ailments (Çaglar et al., 2008; Medici et al., 2005; Urbanska et al., 2009). Health-related benefits of pro-
biotics include antimutagenic, anticarcinogenic properties, enhancement of lactose metabolism, mitigation of intestinal inflamma-
tion and allergic disease control, prevention against diarrhea and infections during pregnancy (Minervini et al., 2017). Probiotics
maintain intestinal flow by enhancing the absorption of iron, magnesium, and calcium. They are also involved in detoxification of
carcinogens and vitamin B production (Granato et al., 2010).
Dairy products can also be used as a probiotic carrier vehicle for other nutritional supplements. For example, a fermented whey
beverage was prepared with the addition of tomato juice (Lycopersicum esculentum). The authors evaluated the characteristics of the
fermented whey beverage with different tomato juice concentrations (5%, 10% and 15%), using probiotic bacteria L. acidophilus and
Lactobacillus plantarum. The authors reported that fermented beverages with 5% tomato juice had the highest overall scores for all the
sensory attributes, including color, texture, aroma, and flavor. The highest antioxidant activity was reported to be in samples con-
taining 15% tomato juice.
Yerlikaya and Ozer (2014) reported a study of probiotic fresh white cheese using co-culture with Streptococcus thermophilus. and
S. thermophilus into milk to produce the probiotic cheese. The effects of the co-culture on cheese composition and microbiological
viability during 28 days of storage were monitored. Sensory characteristics of cheese were studied only on the first and last days of
storage. It was observed that the addition of co-culture did not have any negative impact on the cheese components, including the
properties of fat and dry matter. However, a varying range of pH, salt content, and lactic acid were observed as the probiotic bacteria
population increased (107 CFU/g), higher than the threshold required for probiotic activity. In terms of sensory characteristics, the
highest sensory scores were recorded for cheese inoculated with S. thermophilus and Lactobacillus casei, whereas other probiotic
combinations were less desirable in terms of taste and appearance. In another study, Papadopoulou and Chorianopoulo (2016)
reported the production of a functional, fresh cheese enriched with the probiotic strain L. plantarum T571 which had been isolated
from traditional Greek products. Results indicated that the population levels of LAB surpassed 8 log CFU/g, and by the end of shelf
life, the population levels probiotic were still at close to 7.5 log CFU/g. The product had higher acidity, lower pH and, consequently,
reduced counts of coliforms and Listeria spp. The study implicated the possibility of producing a high value-added fresh cheese
product from a traditional product with enhanced product safety.
Probiotics have been incorporated into frozen desserts such as ice cream. In this process, the fact that oxygen is present during the
freezing step can negatively affect the survival of probiotic anaerobes. A potential workaround to this problem is the use of
6 Probiotics in Functional Foods
micro-encapsulation technologies to protect the probiotic organisms from the detrimental effects of oxygen, as has been demon-
strated in ice cream (Homayouni et al., 2012). The low storage temperature of frozen desserts and ice creams can help to protect
the viability of probiotic organisms and thus ensure large numbers of viable organisms are available at the time of consumption
(Cruz et al., 2009).
Meat Products
In contrast to dairy products, applications of probiotics in meats is rare and still being explored. Traditionally, uncooked fermented
sausages made up of lean meat, fatty tissues, spices, and water-retaining ingredients could be the most common form of probiotics
found in meat products. Sucuk is another type of fermented sausage, originated in Turkey, in which beef is combined with garlic and
pepper and allowed to ferment. In East Asian countries, Nham is one of the meat fermented products, mainly produced from pork
meat wrapped in banana leaves and allowed to ferment for up to 5 days at ambient temperature. Though the spontaneous and
uncontrolled fermentation process is not very well documented, the major microflora found in these traditional products is
a mixture of Lactobacillus sp., Pediococci sp.and Leuconostoc sp. The prevalence of a particular species is highly dependent on the stage
of fermentation and pH of the raw material (Vongsawasdi and Noomhorm, 2014).
Fermented meat products are potentially beneficial to the human gut by inhibiting enterotoxin produced by Staphylococcus aureus
(Sameshima et al., 1998). Researchers have also determined that Lactobacillus rhamnosus GG is suitable as a starter culture in dry
sausages because of its growth characteristics in low moisture environments (Erkkila et al. (2001). In another study, Lactobacillus
sakei C2 has been reported as a starter culture in fermented sausages (Gao et al. (2014). These researchers used isolates of L. sakei
C2 at two different concentrations (105 and 107 CFU/g) during the sausage production. The use of L. sakei C2 as a starter culture
controlled the growth of harmful microorganisms during fermentation. Moreover, with a higher concentration of the starter culture
the authors reported a decrease in malondialdehyde (MDA) and nitrite content as well as a superior flavor and overall acceptability
in the samples of fermented -sausages compared to the control and those processed with a lower concentration of the starter culture.
Overall, the authors observed a significant increase in the color values of L* (lightness), and a* (redness), on inoculation with
L. sakei C2 compared to the control.
Cereal-Based Products
Dairy-based foods containing probiotics provide a number of health benefits to the consumer, but the dairy vehicle is associated
with certain shortcomings for some consumers such as protein allergy, lactose intolerance, and elevated cholesterol content. To
avoid these issues, non-dairy based fermented foods have been developed. Among non-dairy fermented foods, cereal-based fer-
mented foods are gaining popularity due to their wider acceptability (Gawkowski and Chikindas, 2012; Gupta and Abu-
Ghannam, 2012). Some critical differences between dairy and non-dairy based fermented foods are listed in Table 1.
Cereal-based foods are considered to be suitable carriers for probiotics because they address many of the shortcomings of fer-
mented dairy products noted above (Prado et al., 2008). Cereals are also a good source of dietary fiber that can act as prebiotics and
potentiate the growth of LAB (Kumar et al., 2015). In Asia and Africa, the production of fermented cereal-based beverages and
porridges using LAB is one of the oldest known processing techniques. The cereal grains that are commonly used to produce
non-dairy fermented foods include maize, wheat, barley, oats, rye, sorghum, and millet. Whole grain consumption is also known
to reduce the risk of type II diabetes, cardiovascular disease, obesity, and various cancers (Clemens and Pressman, 2006). The
bioavailability of minerals, such as zinc and iron, is increased during the fermentation of cereals. This phenomenon can be
explained by the fact that microbial enzymes such as phytase and production of organic acids during fermentation can potentiate
Table 1 Predominant factors for comparing dairy and non-dairy products con-
taining probiotics (Kumar et al., 2015)
Note: Ve means the parameter has a negative impact of a probiotic product, and þVe means the
parameter indicates the positive potential of the probiotic product. – indicates the parameter has no
effect on the probiotic product.
Probiotics in Functional Foods 7
the liberation of minerals (Hotz and Gibson, 2007). Sorghum flour-based yogurt was recently prepared with acceptable sensory
scores and reported to have a viable count >108 CFU/g, which was higher than the minimum desirable count of 106 CFU/g (Sanni
et al., 2013).
Hua Cui et al. (2013) optimized fermentation conditions of a walnut milk beverage inoculated with kefir grains. The authors
evaluated the effects of fermentation temperature, time, inoculum size, and sucrose concentration on walnut milk beverage fermen-
tation, optimized the fermentation conditions, performed chemical analysis, determined microbial viable cell counts, and carried
out sensory analysis. The authors suggested an optimum fermentation temperature of 30 C, fermentation time of 12 h, inoculum
size of 3 g of kefir grains (wet weight), and sucrose concentration of 8 g/100 mL. Under these optimum conditions, the maximum
sensory score reached a value of 88. Viable cell counts of lactococci, lactobacilli, and yeast sustained in the beverage were 8.2 107,
1.1 108, and 1.0 106 CFU/mL, respectively.
In another study, Lactobacillus amylovorus (DSM19280) was employed in gluten-free sourdough bread to improve the microbial
shelf-life (Axel et al., 2015). The activity of L. amylovorus was investigated as a starter culture to extend the microbial shelf-life of
gluten-free quinoa sourdough bread. Quinoa flour was used for sourdough preparation, and the starter culture was added to the
sourdough to an initial inoculum size of 1 107 CFU/g dough. It was observed that the mold shelf life of the quinoa sourdough
fermented with the antifungal L. amylovorus was extended by 4 days compared to the control (wheat flour). Moreover, bread char-
acteristics such as specific volume and crumb hardness were also improved in the quinoa sourdough, which led the authors to
conclude that L. amylovorus DSM19280 could serve as a potential bio-preservative to produce gluten-free bread with better nutri-
tional value, improved bread quality and shelf-life, and better safety.
Isolation, selection, identification and characterization of probiotic strains are critical steps before utilization and enrichment. The
criteria for probiotic selection include (i) survival through the gastrointestinal tract (demonstrated both in vitro and in vivo); (ii)
demonstrated beneficial effects to the host; (iii) non-toxic; (iv) non-pathogenic; and (v) bioavailability in adequate numbers in
the food matrix to deliver maximum health benefits (Fontana et al., 2013). However, the significance of probiotics should not
be overgeneralized with respect to a wide spectrum of health benefits as these benefits tend to be strain-specific.
The current reliable sources for isolation of probiotic strains include the gastrointestinal tract and human breast milk (Borriello
et al., 2003; Dash, 1980). Research has established that human breast milk can colonize the aseptic intestine of an infant with micro-
flora and contain bacterial strains which can be used as probiotics (Shokryazdan et al., 2014). The several predominant bacteria
present in human breast milk are staphylococci, streptococci, micrococci, lactobacilli, enterococci, lactococci, and bifidobacteria.
Also, probiotic strains such as B. longum and L. acidophilus RY2 have also been isolated from the feces of healthy humans and infants,
respectively (Lin et al., 2009;
Srůtková et al., 2011). Moreover, the guts of several other animal species such as pigs, rats, and poultry
are good sources of probiotics as well (Petrof, 2009). Other potentially rich sources of useful probiotic strains include plant-based
fermented foods, raw milk and fermented foods from animal sources (Fontana et al., 2013). Previous studies have shown that pro-
biotic strains isolated from fermented foods can be used as starter cultures to produce commercial fermented probiotic foods
(Mathara et al., 2008). Although probiotics intended for human use should be of human or human food origin as they are safer
and could attach to human intestinal epithelial cells, they could also be used for animals and poultry (Sanders, 2008).
There are several techniques by which probiotic strains can be identified (Davis, 2014; Yadav and Shukla, 2017). The most
commonly available techniques can be categorized into in vitro screening tests or molecular techniques. These techniques are based
on species-specific identification which include amplified ribosomal DNA restriction analysis (ARDRA), 16S and 23S rRNA
sequencing, and strain-specific identification techniques such as ribotyping, random amplified polymorphic DNA (RAPD),
pulse-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), PCR-denaturing gradient gel electro-
phoresis (DGGE), fluorescent activated cell sorting (FACS), nucleic acid-based enumeration methods (PCR methods), and fluores-
cent in situ hybridization (FISH). Recently, Ngongang et al. (2016) isolated lactic acid bacteria from palm raffia wine and evaluated
the probiotic potential of this isolate on serum lipid cholesterol and enzyme activities in wistar albino rats. The isolates which were
selected and identified included L. plantarum and L. pentosus, which elicited a significant reduction in cholesterol level. L. pentosus
showed a 76% cholesterol reduction compared to L. plantarum, which showed a 52% reduction. Interestingly, L. pentosus also selec-
tively reduced the levels of total cholesterol, Low Density Lipoprotein (LDL), and Very Low Density Lipoprotein (VLDL) in the rat’s
blood serum. These bioactivities suggest that L. pentosus and to a lesser extent, L. plantarum could have potential as novel probiotic
strains.
The industrial production of probiotics depends on two major pre-requisites; the availability of sufficient medium for the pro-
biotic organisms to grow in substantial quantities, and their potential viability. Further, additional production limitations in the
growth of certain strains through the manufacturing process, such as processing parameters and the presence of preservatives,
need to be addressed (Fontana et al., 2013). Probiotics can also be produced by immobilization technologies using microencap-
sulation techniques on biopolymers, alginates, milk, and whey protein gels, and natural supports like fruit and cereal pieces (Mitro-
poulou et al., 2013), and these approaches will be addressed in the following section.
Several animal and human studies have established the apparent beneficial health effects of probiotics (Krumbeck et al., 2016).
However, more research needs to be conducted on the health impacts of probiotics to understand the underlying mechanistic rela-
tionships between fermented foods, probiotics, and human health.
Probiotics are primarily delivered to humans using food vehicles. The food vehicle can serve as a protectant for probiotics transiting
the stomach and gastrointestinal tract (GIT), regulate colonization, and can contain active ingredients that promote the efficacy of
probiotics (Ranadheera et al., 2010). The oral administration of probiotics results in the loss of viable bacteria due to the acidic
environment of the stomach, and this loss of viability results in decreased efficacy of administered probiotics (Cook et al.,
2012). Therefore, for oral delivery via foods and beverages the selected probiotics should exhibit acid and bile stability (Tuomola
et al., 2001), or be adequately protected against the harsh environment in the stomach. In order to establish scientific evidence for
the bioavailability and functionality of probiotics in the human body, several techniques are being used such as GI-tract diagnostics,
immunology, delivery strategy, and biomarkers. This also includes controlled human studies using specific strains of probiotics for
general as well as targeted population groups. Significant loss of efficacy and viability occurs due to unfavorable stomach conditions
including high acidity, low pH and presense of bile salts in the small intestine ultimately reducing the viability of administered pro-
biotics at target sites (González-Ferrero et al., 2018). Such conditions ultimately lead to a compromised competition of probiotics
with endogenous microbiota (Vandenplas et al., 2015). The other contributing factor that decreases the efficacy of probiotics is their
fragile nature as the probiotic viability might have been compromised during processing or storage (Farnworth and Champagne,
2010). The main challenge faced by food manufacturers is to ensure the stability and viability of probiotics in the food product
throughout the shelf life (Mattila-Sandholm et al., 2002). Mattila-Sandholm et al. (2002) listed ten technological factors affecting
the functionality of probiotics, including; stability, non-viability, viability, micro-encapsulation, drying technology, fermentation
Probiotics in Functional Foods 9
technology, targeted probiotics, food matrix formulation, strain characteristics, and daily dosage. Fundamentally, these factors must
be optimized in relation to market demand for a food product. For example, a food product with good sensory properties, phage
resistance, live culture levels of 1010 and above to ensure viability during processing and storage could be a daunting task. Apart
from stability under acid and bile conditions, another important selection parameter for probiotic strains is their ability to adhere
to the intestinal mucosa. The importance of adhesion can be understood by the fact that reasonable numbers of probiotic organisms
may be sufficient to transit from the stomach to the small intestine to eventually grow in the gastrointestinal tract.
The diverse nature of the GI tract presents a number of challenges to the delivery of probiotics at targeted sites. The type and
content of biomacromolecules (fat and protein) in food systems can influence the growth and survival of probiotics. Therefore,
the formulation of the product can be altered to aid probiotic delivery and ultimate efficacy (Ranadheera et al., 2010). Dairy prod-
ucts are considered excellent vehicles for the delivery of probiotics to the human GI tract (Ross et al., 2002) as they increase surviv-
ability of probiotics by providing a buffering environment to stomach acid. For this reason, yogurt and fermented dairy products are
the leading vehicles for the delivery of probiotics. Ice cream and frozen dairy products have great potential as probiotic carriers due
to low storage temperature that ensures the high viability of probiotics at the time of consumption (Cruz et al., 2009). Cheese is
a well accepted and versatile food commodity and serves as a suitable candidate for the delivery of probiotics. As a probiotic carrier,
the consumption of cheese has increased over the past decade (da Cruz et al., 2009). Cheese fortified with inulin and oligofructose
presented better sensory qualities and high probiotic viability.
Growing concern of allergy and lactose intolerance have led to the development of probiotic fortified non-dairy products such as
fruit juices. While dairy products serve as excellent probiotic carriers for most strains, applications of probiotic cultures in non-dairy
heterogenious matrices pose considerable difficulty. As the probiotics viability highly depends on factors such as pH, water activity,
temperature, oxygen tension, strain antagonism, and molecular inhibitors, careful selection of probiotic strains is of the utmost
importance. The cell viability and functionality need to be ensured by adding suitable prebiotics (e.g. GOS, FOS, inulin).
Probably, the most common technique to protect probiotic bacteria is encapsulation. Fig. 4 provides an overview of various
types of encapsulation techniques. Briefly, the technique of encapsulation is the coating of microscopic particles, in this case the
probiotic organisms, with another material (Sun and Griffiths, 2000; Ding and Shah, 2009). The size of the encapsulated particles
often influences the delivery of high numbers of probiotic organisms. Among the encapsulation techniques, extrusion, emulsion
and spray drying have gained industrial acceptance for manufacturing probiotic products. Alginate is one of the most common
encapsulating agents, polysaccharide with linear chains of b-(D)-glucuronic (G) and a-(L)-mannuronic (M) acids, derived from
brown algae or bacterial sources (Rinaudo, 2008). Alginate is commercially available in a wide range of molecular weights ranging
from tens to hundreds of kDa (Thu et al., 1996). Alginate is well suited for probiotic encapsulation due to its mild gelling capacity,
generally recognized as safe status and lack of toxicity (Gombotz and Wee, 1998). Although alginate has been extensively used for
probiotic encapsulation, many other polysaccharides have shown promising results for the encapsulation of probiotics and protect-
ing the strains from the harsh conditions in the stomach and GIT. Common hydrocolloids, such as xanthan gum alginate, among
others, are commonly used for probiotic encapsulation (Garcıa-Ochoa et al., 2000; Tuladhar and Anal, 2014). A summary of
common encapsulation polymers and their significance is given in Fig. 5.
In addition to polysaccharides, proteins have become a valuable alternative for probiotic encapsulation due to their complex
tertiary structures and their emulsifying properties. For example, the emulsification performance of gum arabic can be attributed
to its protein content (2%, w/w). Proteins are also useful in creating a bond between the shell (wall material) and the core (pro-
biotic), due to their inherent gelling properties, and their ability to gel through the use of enzyme derived cross-linkages, heat-
based gel transition or chemical cross-linking. Casein, bovine serum albumin and soy protein are commercially available and
frequently used in probiotic encapsulation (Cook et al., 2012). González-Ferrero et al. (2018) reported the encapsulation of pro-
biotics by using a byproduct, soybean protein concentrates as a wall forming material. They used coacervation of protein by using
calcium salts and spray drying and found that the viability of probiotics (L. plantarum and L. casei) and tolerability to GIT conditions
were improved compared to control experiments.
Proteins are also useful for site targeted delivery of probiotics. Crittenden et al. (2014) reported the targeted delivery of probiotics
encapsulated in oil-in-water emulsion system containing film-forming protein and a carbohydrate. Ying et al. (2013) reported
microencapsulation of L. rhamnosus GG by spray drying. In this work, whey protein isolate and resistant starch were used as the
encapsulation materials. L. rhamnosus was able to survive for 5 weeks in apple juice at storage temperatures of 4 and 25 C. In
a similar attempt to encapsulate L. plantarum in a calcium-alginate-soy protein isolate hydrogel, processing conditions (pH and
temperature) were optimized for the inclusion of probiotics in pasteurized mango juice. Notably, the encapsulated probiotic
bacteria were found alive even after treatment at 72 C for 90 s, while quite obviously, most of the non-encapsulated cells did
not survive at a temperature >50 C. The survival of probiotic cells was found higher with the hybrid hydrogel beads containing
alginate and soy protein isolate (1:8 w/w) (Praepanitchai et al., 2019). The work underlines the importance of encapsulation to
the survival of probiotics during processing and when incorporated into acidic products.
Probiotics in Functional Foods 11
-Fermentation
medium
Fermentation -pH and acidity
conditions -Temperature
-Dissolved
oxygen
Food
ingredients
Encapssulation
Viability of
probiotics in
food
Protective
materials
- Drying
Processing
- Freezing
conditions
-Thawing
Packaging and
storage
conditions
Figure 6 Predominant factors affecting probiotic viability in production and in food systems (Tripathi and Giri, 2014).
12 Probiotics in Functional Foods
This fact can be beneficial for industries that require bacteria with high heat tolerance during processing. The previous reports
revealed that mild heat treatment of Lactococci and Lactobacilli before heat stress showed an improvement in thermal tolerance
(Desmond et al., 2001). During fermentation, the exposure of strict anaerobic bacteria to oxygen results in decreased viability
(Gaudreau et al., 2013). To overcome this issue, several methods have been introduced to reduce the oxygen content during
fermentation, and the most important one is vacuum fermentation (da Cruz et al., 2007).
The protection of probiotics at non-refrigerated temperatures is still a challenge for food processing and the incorporation of
probiotics into shelf-stable products. Crittenden et al. (2006) reported the protection of probiotics during non-refrigerated storage
by encapsulation of probiotics in an emulsion system, comprised of protein, carbohydrate and lipid film. The researchers attempted
to form a synbiotic combination with Bifidobacterium infantis strain and the formulation of a film-forming protein-carbohydrate-
lipid emulsion. This novel approach produced shelf-stable microcapsules with lower water activity (<0.3) making it possible to
store at ambient storage temperature. Moreover, the release of bacteria in simulated stomach conditions was found to be rapid
and showed increased tolerance to acidic conditions. The protein-carbohydrate–lipid matrix proved to be a better alternate to algi-
nate, carrageenan, and modified starches due to their sensitivity to the acidic environment. Other approaches include formulating
an aqueous suspension or dispersion of a protein and carbohydrate, and oil in water emulsions to which the probiotic is introduced
as a liquid media. The suspension, dispersion or emulsion are subsequently freeze-dried (Crittenden et al., 2005)
Weinbreck et al. (2010) determined the viability of probiotic Lactobacillus rhamnosus GG (LGG) at medium to high levels of water
activity in an accelerated shelf-life study at 37 C. A high water activity (0.7) was found to be detrimental to the survival of unen-
capsulated LGG, yielding more than a 10 log loss in cell viability within 2 weeks. Vesterlund et al. (2012) reported the influence of
a range of water activities (0.11, 0.22, and 0.43) on flaxseed incorporated with Lactobacilus rhamnosus GG, which was found to be
highly depended on water activity. Up to 14 months of storage at ambient temperature was achieved. While encapsulation is a viable
option to maintain probiotic populations in low and intermediate-moisture environments, incorporation of probiotics to low or
intermediate-moisture content foods is not without challenges. On one hand, a food product with a water activity <0.6 is generally
considered shelf-stable, low levels of available moisture adversely affect the viability of some probiotic strains. Therefore, dehydra-
tion of microencapsulated probiotics requires careful consideration of the most suitable technique. These findings indicate impor-
tant implications for the snack and energy/meal bar food categories. As stated above, encapsulation is considered a handy tool for
preserving probiotic viability in dry and semi-dry products, research is continually evolving to validate the approach at different
shelf-life requirements, moisture contents, and food product matrices. Weinbreck et al. (2010) noted that there is no “no quick-
fix” for maintaining cell viability under low moisture conditions. Although the encapsulation techniques provide some shelf-life
extension, a better approach to ensuring probiotic survival requires “a multifaceted approach” by utilizing probiotic genotypes
and phenotypes to improve and maintain probiotic survival in a dried shelf-stable food matrix.
One such study was conducted by Ying et al. (2013) on survival of spray dried microencapsulated Lactobacillus rhamnosus GG
(LGG) added into apple juice or citrate buffer (pH 3.5) and stored at 4 or 25 C over a 5-week period. The study utilized whey
protein isolate modified resistant starch at various ratios. The combination with resistant starch and whey protein provided better
protection to LGG in naturally acidic apple juice due to the ability of the whey protein isolate to create a buffered microenvironment
within the hydrated colloid particle surrounding the bacterium.
Frozen food products can ensure the long-term survival of probiotics due to low temperatures. However, freezing can damage
the probiotic cell membranes due to ice crystals, and this can lead to probiotic cell death. During the freezing process, cells are dehy-
drated due to solute condensate that results in interruptions of vital metabolic activities (Akın et al., 2007). The rate of freezing also
plays an important role, as a slow freezing rate produces large ice crystals that are lethal for bacterial cells, and rapid freezing
promotes better survival of probiotics in the food product (Gill, 2006; Mohammadi et al., 2011). During thawing, probiotic
viability can also be decreased due to physical phenomena such as osmotic effects, the presence of a high concentration of hydrogen
ions and organic acids in the food matrix (Jay et al., 2005).
Probiotic products are often dried at ambient temperature to increase the shelf life and reduce the costs associated with freeze
storage. Drying facilitates ease of probiotic storage and transport. However, probiotic product drying is one of the most significant
challenges for food manufacturers as it reduces the viability of cells (Santivarangkna et al., 2006). Food processors employ various
techniques for drying such as hot air drying, spray drying, vacuum drying, freeze-drying, and related techniques. For liquid food
products, spray drying is the most economical method, but it leads to a decrease in probiotic viability due to high temperature,
dehydration, and osmotic effects.
By comparison, freeze-drying maintains the viability of probiotics, but at the same time, freeze-drying is an expensive method.
Recently, fluidized bed drying and radiant energy vacuum drying techniques were reported to improve the probiotic viability
compared to other drying techniques (Nag and Das, 2013; Noorbakhsh et al., 2013). Other low-temperature drying techniques
not mentioned above include; ultrasonic vacuum spray drying, spray chilling, electrospinning, supercritical, and enzyme gelation.
Since spray drying is the most practical approach for granulation, it is widely employed by the nutraceutical industry. However, as
mentioned earlier, one way of protecting probiotics is the use of heat resistant starches, maltodextrin and whey proteins as coating
materials (Behboudi-Jobbehdar et al. (2013). A combination of heat resistant encapsulation materials, flow rate and temperature
(133.34 C and 7.14 mL/min), production of viable L. acidophilus was maximized. Semyonov et al. (2011) have reported achieving
low-temperature spray drying of probiotics in two stages; vacuum spray drying of the solution followed by fluidized-bed drying of
the powder.
Regardless of the dehydration techniques, the dehydrated probiotic products are hydrated to revive the cells. Freudig et al. (1999)
reported that the rehydration process is categorized into four steps: wetting, submersion, dispersion, and dissolution. Among these
Probiotics in Functional Foods 13
steps, wetting and rehydration time are controlling factors as the volume of rehydration solution influences the recovery of probi-
otics to viable states (Vega and Roos, 2006). Teixeira et al. (1994) reported that slow rehydration provides optimum results for
dehydrated recovery cells. Saccharomyces cerevisiae presented better viability and recovery when the dried cells were rehydrated slowly
(7–16 days) under controlled conditions (Poirier et al., 1999). However, due to costs associated with such a long rehydration
process, the approach has not gained popularity.
Novel non-thermal processing technologies are seen as an alternative to traditional thermal processing to enhance the delivery of
functional components produced by probiotics. These technologies include ultrasound processing (Camiti et al., 2011), ohmic-
heating, radio-frequency or microwave heating, high hydrostatic pressure processing, radiation processing (Carocho et al.,
2013), and pulsed electric field processing (Bi et al., 2013). A detailed review of these processes and their role in the bioavailability
of functional components has been compiled by Jongyingcharoen and Ahmad (2014). While the body of knowledge for the
survival of probiotics under these novel processes is continuously evolving, enough evidence is available for the retention of pro-
biotics and functional components. The applicability of these technologies is subject to further research and for the optimization of
processes and nutritional retention in food products. More recently, hybrid systems of thermal and non-thermal processes have
been seen as a considerable potential to reduce processing impact on functional foods (IFT, 2017).
The food industry is witnessing a shift from traditionally fermented products; dairy, pickles, and beverages to a multibillion-dollar
industry of health foods specially designed to cater to the needs of specific and targeted population groups. Probiotics and other
active compounds, once marketed as medicinal and dietary supplements, have now moved mainstream under the umbrella of
health foods. The health food category of specialty foods continues to be among the top food trends over the past several years.
However, the mainstreaming of probiotic-rich food is not without its challenges. From the manufacturing perspective, the techno-
logical requirements of probiotic strains for both pharmaceuticals and nutraceuticals are different. The former requires dosage accu-
racy, and the latter requires a sound sensory outlook of the product. The most challenging step in the delivery of probiotics is to
maintain their viability in the food product at the time of consumption and to enhance bioavailability in the human gut. Despite
the vast body of literature produced over the years, developing a food matrix enriched with probiotics is still a subject of interest for
research concerning their interaction with proteins, carbohydrates, and flavoring agents as they have shown to modify probiotic
efficacy and viability. Therefore, the food industry is adopting a number of strategies to ensure the viability of probiotics during
production, processing, and storage of food products in addition to focusing on probiotic strains. Among them, microencapsulation
and dehydration of probiotic microcapsules are popular ways to improve the microbial viability during both storage and in the
gastrointestinal tract. To ensure probiotic delivery, a sound quality assurance program is needed for both direct determinations
of successful probiotic delivery or indirect estimation, such as physicochemical properties (adhesion to the human mucous
membrane to colonize the gastrointestinal tract). Modern tools of molecular informatics could be beneficial in achieving this
goal. To this end, a considerable opportunity exists in exploring the idea of a “personalized microbiome” which is expected to domi-
nate future market trends. Finally, the onus is on the manufacturer to maximize health benefits to consumers, which can be achieved
by selecting the right strains and culture conditions, optimizing processing conditions, and employing innovative techniques.
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