This document reviews strategies for producing probiotic non-dairy products and evaluating their health benefits. It discusses using fruit juices as a matrix for probiotic microorganisms and describes studies processing probiotic mango and mixed berry juices. Major factors affecting probiotic survival in juices are addressed, along with strategies like fortification with prebiotics, microencapsulation, adaptation, and continuous fermentation technologies. The viability of probiotics in food products depends on maintaining concentrations of 106-107 CFU/g at consumption.

1. Arba Minch University
College of Natural Science
Department of Chemistry
Production of non - dairy probiotic product and evaluation of their
health benefits:
A Review
By
Aschalew Demssie
Advisor
Dr. S. Babuskin
Arba Minch, Ethiopia
October 2018
1

2. INTRODUCTION- Probiotics
Probiotics are live microorganism that confer health benefits on the host when
administered in required amount (WHO/FAO, 2002).or
Probiotics are friendly bacteria, or good bacteria already found in our body,
particularly those in our gut.
According to different studies, the normal human intestinal tract contains 300-1,000
different kinds of bacterial species with about 1014 individual bacteria
The human large intestine bacterial count reaches up to 1012 Cfu/g, 108 Cfu /g in
the small intestine,
but only 102 CFU /g in the stomach due to its low pH.
2

3. Conti…
In the eyes of human beings, microorganisms are disease causing organisms.
However, based on fact, only a small fraction of the total microbial populations are
associated with disease production, and the remaining is beneficial to human beings.
These tiny organisms, with sizes ranging from less than a micrometer to a few
micrometers, are sometimes called small chemical factories because of their ability
to produce a variety of chemical substances that are useful for us.
3

4. Conti..
The most commonly used probiotic bacteria are Lactobacillus sp. and
Bifidobacterium sp.
These genera are the main inhabitants (colonize) of the human intestine.
For example, Bifidobacterium inhabits the large intestine where as Lactobacillus
inhabits the small intestine.
The fermentative nature and synergism property of these bacteria make them suitable
to be used as probiotics.
They are general recognized as safe (GRAS) with no pathogenic, or virulence
properties have been reported.
4

5. Conti..
The adult intestinal tract is a fairly stable microbial ecosystem and contains a balance of
beneficial and pathogenic microbes.
However, this balance is disrupted by an increase in pathogenic bacteria due to conditions
such as:
Antibiotics and other drug intake.
Microbial infections
Diet(highly processed, low fiber foods)
Chronic diarrhea
Stress
Chlorinated water
Radiation and chemotherapy
age
5

6. Conti…
When we take antibiotics, they can disrupt the bacterial balance by not only killing
the bad bacteria in the GI tract, but by also wiping out the beneficial bacteria.
when bad bacteria have overgrown in our GI tract and can cause illness.
Therefore, probiotics help restore a healthy balance by adding “good” bacteria back
to the gut and reducing the growth of any “bad” bacteria .
Improving the digestive system, and keep the over all health of individual.
6

7. Conti..
Thus, Probiotics are offering tremendous health benefits to the consumers,
such as:
the improvement of gut health by balancing the equilibrium.
boosting up of the body’s natural defense mechanisms
Control of Inflammatory bowel diseases
Control of irritable bowel syndrome (chronic functional disorder of the colon).
Alleviate food allergy.
Enhancing metabolic process
Suppression of exogenous and endogenous pathogens.
Improvement in lactose intolerances.
7

8. fruit juices matrix for probiotic microorganisms
Dairy fermented products have been considered as the best carriers for probiotics;
but, according to research reports about 75% of the world population is affected by
lactose-intolerance. (Reference)
Furthermore, the use of milk-based products may be also limited by:
allergies
cholesterol diseases
dyslipidemia (unhealthy level of lipids).
Now a day, Increasing the demand of vegetarianism.
8

9. Conti..
Recently, beverages based on fruits, vegetables, cereals, and soybeans have been
proposed as new products containing probiotic strains;
particularly, fruit juices have been reported as a novel and appropriate medium for
probiotic because of their essential nutrients content.
Numerous researchers reported on the beneficial health effects of juices; for
example, aqueous extracts of kiwifruit and avocado had very less cytotoxicity plus
high anti-inflammatory activity (Sutton, 2007).
9

10. Conti…
Fruit juices offer several advantages as probiotic vehicles.
They are a rich source of nutrients (antioxidants, minerals, and vitamins), and their
natural sugars contribute to the probiotics growth.
Moreover, the fruit juices also have a good refreshing taste and are a consumption
choice for people of all age groups.
Another advantage is that for these juices digestion in the stomach is faster than that
for dairy products.
Thus, the microorganism spends much less time in the acidic environment of the
stomach.
10

11. Conti…
While looking for different food matrices many researchers have been
investigated the suitability of various fruit and vegetable juices, such as:
avocado
tomato
mango
orange apple
grape
Peach
pomegranate
Watermelon
Carrot
beet root and cabbage juices as raw material for the production of probiotic
juices or related beverages.
11

12. Conti..
Several studies demonstrated the positive effect of the intake of nine micronutrients
that can be easily found in fruits such as:
 calcium, foliate
 retinol, nicotine acid
 vitamin E, riboflavin
 β-carotene and biotin, pantothenic acid
Furthermore, fruit juices have shown negative effects on some pathogenic
microorganisms, conversely improves the growth of beneficial bacteria.
These properties of fruits and vegetables attributed the survival and cell viability of
probiotics in food products and provide additional health benefit for consumers.
12

13. Review of Literature
13

14. Processing of probiotic fruit juices- Mango Juice
 There are different fruits known to produce probiotic fruit juices, among these
mango juice is the promising carrier for the production of novel probiotic products
(reference).
 The probiotification of mango juice was carried out by lactic acid bacteria
fermentation.
 Mango juice fermentation was performed at 30 ºC for 72 h under micro - aerophilic
conditions.
 The microbial population, pH, titrable acidity, sugar, and organic acid metabolism
were measured during the fermentation period and the viability of the strains was
determined under the storage conditions at 4 ºC for 4 weeks.
14

15. Conti…
 The lactic acid bacteria reduced the pH to as low as 3.2 from 4.5 within 72 h of
fermentation,
 And the substrate concentration was reduced to 5.8% (w/v) from 12% (w/v).
 Lactobacillus plantarum exhibited the fastest utilization of sugar and reduction of pH
in the mango juice when compared to the other strains used.
 The viability of the cells was maintained at 1.0 × 107 CFU/ml throughout the storage
period.
 From this investigation, it can be concluded that mango juice is suitable for the
production of probiotic beverage .
15

16. Conti…
Processing of probiotic fruit juices- mixture of juice by lactic acid bacteria, including
Lactobacillus casei and Lactobacillus acidophilus
 For producing probiotic mixture of Barberry and Black cherry juice microbial
suspension with an initial concentration of 107 CFU/ml was prepared.(Reference)
 Various ratios of bacteria including
 (65% Lactobacillus casei + 35% Lactobacillus acidophilus)
 (50% Lactobacillus casei + 50% Lactobacillus acidophilus)
 (35% Lactobacillus casei + 65% Lactobacillus acidophilus) were inoculumed to 5%
and 10% concentrations of mixture of Barberry and Black cherry juice with 0.2%
whey powder.
16

17. Conti…
 Fermentation process was carried out for 72 h at 30 °C.
 Microbial growth, changes in pH, lactic acid and reducing sugars were analysed after
fermentation, and also viability of probiotic bacteria, changes in pH and lactic acid were
determined during 28 days storage at 4 ⁰C.
 According to the obtained results, treatment samples(35% Lactobacillus casei + 65%
Lactobacillus acidophilus) with 10% concentration, was detected as the best treatment,
because it had the maximum bacterial growth and cell viability, the minimum pH and the
maximum amount of lactic acid.
 It has been observed that, the mixture of Barberry and Black cherry juice with 0.2% whey
powder could be considered as a suitable matrix for growth of probiotic bacteria and
functional beverage production.
17

18. Development of probiotic vegetable juices
 The suitability of carrot juice for the production of probiotic food with L. acidophillus, L.
plantarium, L. casei and Bifidum longum was investigated. (Reference)
Response surface methodology (RSM) was employed to analyze the effect of independent
variables (temperature and pH) on response variables (biomass and cell viability).
 The statistical analysis revealed that the optimum pH for probiotic carrot juice production
was 6 and optimum fermentation temperature 30°C.
 During the study of growth kinetics, gradual change in pH, acidity and sugar concentration
was observed which indicates the growth of probiotics. The results suggest that fermented
carrot juice can serve as a suitable media for the growth of probiotics.
18

19. Major Selection criteria of Probiotics strains
Several aspects, including safety, functional, technological and physiological characteristics have to
be taken into consideration during the selection process of probiotic microorganisms.
19

20. Major factors affecting probiotic survival in fruit juices
The health benefit of probiotics mainly relies upon
 Their concentration in foods (CFU/ml).
 plus on their ability to survive the unfavorable conditions of the gastrointestinal
tract.
 Maintaining the viability (at least 106
- 107 CFU/ml) at the end of shelf-life.
 the major influencing parameters can be:
intrinsic food parameters: pH, titratable acidity, molecular oxygen, water activity,
presence of salt, sugar and chemicals like hydrogen peroxide, bacteriocins, artificial
flavoring and coloring agents.
20

21. Conti…
 processing parameters:
extent of heat treatment
incubation temperature
cooling rate of the product
packaging materials and storage methods
scale of production
 microbiological parameters:
 kinds of probiotic strain
 inoculum proportion and rate
Compatibility of different strains
21

22. Strategies to improve survival of probiotics in juices
Fortification with Prebiotics:
The most attractive way to improve probiotic stability in fruit juice could be
fortification with some prebiotics such as:
 dietary fiber
 cellulose or with some ingredients able to exert a protective effect within the fruit
juice.
 Eg. Researchers fortified juices with 20% of β-glucan and demonstrated that in
apple juice, oat flour could protect Lb. rhamnosus during refrigerated storage.
22

23. Conti…
prebiotics, typically non-digestible fiber
compounds, act as food to stimulate the growth
and/or activity of beneficial bacteria (such as
Bifidobacteria or Lactobacillus
Natural dietary sources of prebiotic compounds
include fiber rich foods such as vegetables,
fruits, legumes, and unrefined grains (bran).
As a functional component, prebiotics are often
added to foods such as cereals, biscuits, breads,
table spreads, drinks,
23

24. Conti…
The most common prebiotic compounds include:
 inulin,
 fructooligosaccharides (FOS)
 galactooligosaccharides (GOS).
It has been shown that certain prebiotics, consumed in adequate amounts,
can improve digestion,
prevent constipation,
inhibit growth of pathogenic bacteria,
improve insulin and lipid metabolism,
improve absorption of certain minerals such as calcium.
24

25. Microencapsulation
it is important for the survival of probiotics during storage and its passage through the
digestive tract.
Materials used to encapsulate probiotic cells include different polysaccharides such as
alginate, plant/microbial gums, starch, cellulose.
This carrier material must be food grade if used in food industry, and able to form a
barrier to protect the encapsulated substance.
Probiotic cells have successfully been microencapsulated to preserve them from
detrimental factors during processing and storage such as low pH and high acidity.
It could be concluded that microencapsulation improved the survival of probiotic
bacteria when exposed to acidic conditions, bile salts, and mild heat treatment.
25

26. 26

27. Adaptation and Induction of Resistance
According to several authors, the exposure of probiotic strains to a sub-lethal stress
could induce a sort of resistance and an adaptive stress response.
Researchers successfully evaluated the viability of probiotics in orange, pineapple,
green apple, and red fruit juices and observed strong loss of probiotics viability in
red-fruit juice, due to a combined effect of low pH and phenols.
Consequently, authors used two different strategies:
growth of strain in a lab medium containing different amounts of red fruit juices
added with vanillic acid (phenol stress) or acidified to pH 5.0 (acid stress).
These approaches resulted in a prolongation of the probiotic viability by 5 (phenol
stress) or 11 days (pH stress).
27

28. Fermentation technologies to enhance cell viability
 probiotic production has almost exclusively been carried out using conventional
batch fermentation and suspended cultures, in some cases combined with the use of
sub lethal stresses to enhance cell viability.
 However, other less conventional fermentation technologies, such as continuous
culture and immobilized cell systems, could have potential for enhancing the
performance of these fastidious organisms.
 These technologies can be employed to develop strains with improved physiology
and functionality in the gut and to enlarge the range of commercially available
probiotics, as well as expanding product applications.
28

29. Conti…
Batch fermentation involves: growing a microorganism in the presence of a fixed
amount of substrate and nutrients, added at the beginning of the fermentation
process.
Batch cultivation may have the disadvantage of low productivity, and long turn-
around times, required for cleaning and sterilization of the bioreactors.
29

30. Conti…
Continuous fermentation technology involves: operating fermentation at a specific
growth rate of the organism by controlling substrate input and product output rate.
The main advantage of this type of fermentation is high productivity,
but it is more susceptible to contamination, and cell characteristics can be lost over
time .
Therefore, Under carefully selected conditions, it can lead to both, high cell yield and
process volumetric productivity, as well as decreasing the demand for downstream
processing capacity.
30

31. Conti…
Eg. two-stage continuous fermentation process for the production of stress-
adapted probiotics, could provide a new tool for the more efficient screening of
sublethal stresses than conventional batch cultures, because the system
stabilizes rapidly to changing conditions in the second reactor (after 5 to 7
residence times, corresponding to a few hours). (reference)
31

32. Conti…
Fermentation of probiotics with immobilized cell technology: it has been used to perform
high cell density fermentations for both cell and metabolite production.
 Several studies have shown that cells produced using immobilized-cell technology have
altered physiology and morphology compared with cells produced during free-cell cultures.
 For example, a shift in the metabolic pathway from homo fermentative to hetero
fermentative was observed during continuous cultures with immobilized Lactobacillus.
 Moreover, several authors have observed an increased tolerance of immobilized cells to
different compounds.
 For food applications, cell entrapment in food grade biopolymer gel matrices (e.g alginate )
has been most widely used.
32

33. Conti…
Several advantages over free-cell fermentations have been demonstrated:
high cell densities.
reduction of susceptibility to contamination,
improved resistance to contamination and bacteriophage attack.
enhancement of plasmid stability .
prevention from washing-out during continuous cultures, and the physical and
chemical protection of cells.
cell concentrations typically ranging from 5 × 1010 to 5 ×1011 colony-forming
units/ml, which are 10 to 50 fold higher than traditional batch cultures.
33

34. Conti…
 Eg. Continuous fermentation with B. longum immobilized in gellant gum gel beads
produced high cell concentrations and 4- fold increased volumetric productivity at a
dilution rate of 0.5 h-1 when compared with free-cell batch cultures at optimal pH.
(reference)
34

35. Conclusions
It can be understood that Fruit juices and related beverages represent a suitable carrier for
the delivery of probiotics. Since, fruits are naturally rich in essential macro- and micro
elements.
The medicinal efficacy of probiotic food products depends upon the number of viable and
active cells per gram of the products, just at the moment of consumption; at least 106 – 107
CFU/g.
The factors that influence selection criteria and the best strategies that improve the survival
of probiotics (microencapsulation, fortification with prebiotics) and new fermentation
technology (immobilized cell culture) stated in earlier studies are some of the important
parameters considered when developing probiotic fruit juice in future
35

36. 36