Field of the invention
The present invention relates to a process for preparation of probiotic fennel (Saunf).
More particularly, the present invention relates to a process for stabilizing the bacteria
using encapsulation and a sequence of regulated process which can be further taken for
coating the fennel seeds.
Background of the invention and prior art
Probiotic fennel has been prepared as a non-dairy product format for delivery of
probiotics for various health benefits. Probiotic bacteria were coated over the fennel
along with sugar syrups and binders. The problem addressed in the present invention
includes the process for stabilization of probiotic bacteria by encapsulating (whey
protein/alginate) it, so that it is able to sustain high temperature conditions used for
coating the fennel granules. The encapsulated bacteria showed good temperature stability
and were then incorporated as coating of fennel.
The process of coating fennel seeds includes stringent procedures in which non-spore
forming bacteria cannot sustain. And majority of functionally active bacteria (probiotics)
are non spore forming. The present process includes a process for stabilizing the bacteria
using encapsulation which can be further taken for coating the fennel seeds. Coated
fennel itself is a novel formulation for delivering probiotic bacteria for various benefits
including gut and oral health.
According to Kaila Kailasapathy; "Microencapsulation of Probiotic Bacteria: Technology
and Potential applications"; Curr. Issues Intest.Microbiol.; (2002) 3: 39-48, many reports
indicated that there is poor survival of probiotic bacteria. Providing probiotic living cells
with a physical barrier against adverse environmental conditions is therefore an approach

currently receiving considerable interest. The technology of microencapsulation of
probiotic bacterial cells evolved from the immobilized cells culture technology used in
the biotechnological industry. Several methods of microencapsulation of probiotic
bacteria have been reported and include spray drying, extrusion, emulsion and phase
separation. According to the publication, entrapment of cells in a gel matrix of alginates
is the most popular system of immobilization.
Further according to Mokarram, R.R, et al., "The influence of multi stage alginate coating
on survivability of potential probiotic bacteria in simulated gastric and intestinal juice";
Food Research International; 42 (2009); 1040-1045, probiotics defined as "living
microorganisms which upon ingestion in certain numbers, exert health benefits beyond
inherent general nutrition" have become increasingly popular during the last decade.
Several factors have been claimed to affect the viability of probiotic cultures including
temperature, pH. Alginates are natural hetero polysaccharides made up of D-mannuronic
and L-guluronic acid residues joined linearly by (l-4)-glycosidic linkages. The use of
alginate is favored because of its low cost, simplicity and biocompatibility.
In addition, WO2011/110926 relates to a food product comprising probiotic bacteria
coated with a coating of vegetable origin. In particular, it relates to a dairy product
selected from among milk, cheese, butter, margarine, yogurt and cream comprising
probiotic bacteria coated with at least one coating of vegetable origin.
Although encapsulation of bacteria for providing shelf stability is known in the art, the
present invention relates to encapsulation of bacteria is only a step in the process for
preparation of coated fennel.
However, there remains a need to be able to prepare a dairy food product containing
probiotic bacteria, in which the initial quantity of bacteria present is not subject to an
excessive decline over time leading to a drastic reduction therein. Finally, it is necessary
for the food product containing the probiotic bacteria to be prepared in such a way as to
maintain the bacteria in a good state of vitality and functionality.

None of the prior arts teach a process for the coating a food/seeds with the sugar syrup
containing probiotic bacteria.
Object of the invention
It is an object of the present invention to overcome the drawbacks of the prior arts.
It is another object of the present invention to provide a shelf stable format for delivery of
probiotic bacteria.
It is yet another object of the present invention to provide bacteria stability against hyper
thermal conditions used for coating fennel seeds with sugar syrup an additional step of
alginate encapsulation has been introduced into the process.
Summary of the invention
It is an aspect of the present invention to provide a process for the preparation of
probiotic coated fennel seeds, said process comprising the steps of:
i) Encapsulation of bacteria;
ii) Preparing primary coating comprising steps of:
a) coating fennel seeds by gradational addition of sugar syrup;
b) incubating the said coated fennel seeds at a temperature of 45-55°C for 1-3
hrs
iii) preparing secondary coating comprising steps of:
a) Coating pre-coated fennel seeds with gradual addition of sugar syrup at a
temperature of 55-65°C for 2 hours, wherein said sugar syrup is
supplemented with encapsulated the bacteria,
b) Drying said coated fennel seeds at a temperature of 45-55°C for 2-3 hour.

Brief description of the accompanying figures
Figure 1 illustrates Thermal stability of probiotic bacteria (at 65°C)
Figure 2 illustrates Stability of encapsulated bacteria at high temperature (65°C)
Figure 3 illustrates matrix and shelf stability of bacteria in fennel samples
Figure 4 illustrates effect of gastric conditions on survival of bacteria
Figure 5 illustrates effect of intestinal conditions (bile salts and pancreatin) on survival of
bacteria
Figure 6 illustrates antimicrobial ability of strains using a) Spot on lawn assay (i) and
agar well diffusion assay (ii)
Detailed description of the invention
The present invention relates to a process for the preparation of probiotic coated fennel,
as a shelf stable format for delivery of probiotic bacteria. The present process includes a
process for stabilizing the bacteria using encapsulation which can be further taken for
coating the fennel seeds. Coated fennel formulation for delivering probiotic bacteria
provides various benefits including gut and oral health. For providing bacteria stability
against hyper thermal conditions used for coating fennel seeds with sugar syrup an
additional step of alginate encapsulation has been introduced into the process. The
present invention provides a process for the preparation of a probiotic bacteria coated
fennel seeds that can be used to deliver the microbes for health benefiting potential with
respect to gastrointestinal, immune, oral or metabolic health.
Hyperthermal conditions are used for coating the fennel seeds, as the sugar syrup (used in
formulation for coating) solidifies at low temperature. Thus, the bacteria intended to be
used in the coating should be stable to high temperature conditions. Most of bacteria,

specifically functionally active microbes (Lactobacilli or bifidobacteria) are temperature
sensititive. Thermal stability of in-house screened probiotic strain {Lactobacillus casei
was evaluated at 65°C for 2 hr (Fig 1.). Briefly, 10mg freeze dried Lactobacillus casei
was suspended in 10 ml MRS broth and was incubated at 65 °C water bath for 2 hr.
Bacterial viability was determined by plating the appropriately diluted suspension on
MRS agar.
ITC-6 is Lactobacillus casei strain (ATCC SD5213) and En-Lactobacillus casei refers to
alginate encapsulatcd-Lactobacillus casei.
According to figure 1, there is a drastic reduction of bacterial count when incubated at
high temperature during the course of time. The bacterial viability was observed
atl05cfu/ml. Initial bacterial count of I05cfu/ml was used for evaluation of thermal
stability (65°C) and during the course of incubation drastic reduction in bacterial count
was observed and, after 2hr complete loss of bacterial viability was observed.
Process for encapsulation of bacteria and evaluation of heat stability
Process for encapsulation of bacteria
Probiotic bacteria were encapsulated using whey-sodium alginate solution. Encapsulating
mixture was prepared by using whey protein isolate (62 ml of 11%) and sodium alginate
solution (38ml of 1.5%). One ml of probiotic bacteria (1010cfu/ml) was mixed with 5 ml
encapsulating mixture and beads were prepared in chilled calcium chloride solution using
25G needle.
Evaluation of heat stability:
Thermal stability of encapsulated beads was evaluated at 65°C temperature by incubating
for different time points and bacterial viability was evaluated by platting on MRS agar
plates.
Figure 2 showed a very good thermal stability at 65°C for encapsulated bacteria as
compared to un-encapsulated bacteria (Lactobacillus casei).
Preparation of probiotic fennel:

Step 1: Process for encapsulation of bacteria
Probiotic bacteria were encapsulated using whey-sodium alginate solution. Encapsulating
mixture was prepared by using whey protein isolate (62 ml of 11%) and sodium alginate
solution (38ml of 1.5%). One ml of probiotic bacteria (1010cfu/ml) was mixed with 5 ml
encapsulating mixture and beads were prepared in chilled calcium chloride solution using
25G needle. Encapsulated bacteria (10n cfu for lKg fennel) were then used in
formulation for coating of fennel.
Step 2: Process for coating probiotic fennel
Fennel seeds were coated with sugar syrup in a two step process. For primary coating
fennel seeds (l-2kg) were coated by gradational addition of sugar syrup (500g; 60-70%
sucrose and gum Arabic as binder at 60°C) in a rotating pan. Following which the coated
seeds were incubated at 50°C for 2 hr for reduction of moisture content. Secondary
coating includes addition of probiotic bacteria. For secondary coating pre-coated fennel
seeds were coated with gradual addition (finished in 2 hr temperature 60°C) of 650g
sugar syrup (77% for 1 Kg ) supplemented with 50ml encapsulated bacteria (1010cfu/ml),
in a rotating pan. Afterwards coated fennel seeds were dried at 50°C for 1 hr.
Evaluation of matrix and shelf stability:
For evaluation of matrix stability lg coated probiotic fennel was suspended in MRS broth
and then crushed using micro-pestle, to remove the coating. The suspension was then
plated on MRS agar to enumerate bacterial viability.
For shelf life studies probiotic fennel was aliqouted and stored at room temperature
(ambient conditions) and under accelerated conditions (85 % relative humidity, 37°C).
Samples were taken at different time points (7, 14, 30 days) and processed in MRS broth
for evaluating bacterial viability as described before.
The bacteria was found to be shelf stable upto six months under as demonstrated in fig 3.
Since, one month under accelerated conditions is equal to three month under ambient
conditions.

Gastrointestinal tract stability
i) Stability in Gastric Acid: The bacterial stability was studied in simulated gastric
conditions (gastric juice (saline 0.5%; pH adjusted to 2 with 1M HC1, 3g/lit pepsin)and at
pH2.
According to figure 4, the tested strain, Lactobacillus casei showed stability in gastric
simulated conditions with no significant change in number (p>0.05); thus showing that
the bacteria have a potential to sustain harsh gut conditions.
ii) Stability in intestinal bile: Bacterial stability was also tested in simulated intestinal
fluid (0.5% saline; pH 7.5, 10g/1 bile andlg/1 pancreatin), the conditions mimicking
intestinal conditions in vitro.
According to Figure 5, Lactobacillus casei showed stability in simulated intestinal
conditions with no significant change in number (p>0.05); thus showing that the bacteria
have a potential to sustain harsh gut conditions.
Antagonizing gastrointestinal pathogens (S. aureus and S. typhi))
Antimicrobial potential of lactobacillus strains was evaluated using spot on lawn
assay, using S. aureus and S. typhi as test organisms.
• Streptococcus aureus: Gram +ve: causes gastroenteritis
• Salmonella typhi: Gram -ve; causes enteric fever
According to Figure 5, selected probiotic strain demonstrated good antimicrobial activity
against S. aureus (gram positive) and S. typhi (gram negative).
Fennel seeds were used for delivery of probiotic strain (Lactobacillus casei) established
in vitro to have beneficial effect on gastro-intestinal health, with the ability to sustain in
gastrointestinal tract and inhibition of gastro-intestinal pathogens.

WE CLAIM:
1. A process for the preparation of probiotic coated fennel seeds, said process
comprising the steps of:
i) encapsulation of bacteria;
ii) preparing primary coating comprising steps of:
a) coating fennel seeds by gradational addition of sugar syrup;
b) incubating the said coated fennel seeds at a temperature of 45-55°C
for 1-3 hrs
iii) preparing secondary coating comprising steps of:
c) coating pre-coated fennel seeds with gradual addition of sugar syrup at a
temperature of 55-65°C for 1-3 hours, wherein said sugar syrup is
supplemented with encapsulated the bacteria,
d) drying said coated fennel seeds at a temperature of 45-55°C for 2-3 hour.

2. The process as claimed in claim 1, wherein the sugar is selected from sucrose,
lactulose, invert sugar, dextrin, sucrolose, aspartame, xylitol, sorbitol, mannitol,
malditol or combination thereof.
3. The process as claimed in claim 1, wherein the said binder component is selected
from gum arabic, starch/flour, guar gum, pectin, gelatin, alginates, carrageenan,
gellan gum, gum ghatti, oat gum, xanthan gum or their combination.
4. The process as claimed in claim 1, wherein the bacteria is encapsulated with
alginate.
5. The process as claimed in any of the preceding claims, wherein the said
encapsulated bacteria has a thermal stability at 65°C.
6. The process as claimed in any of the preceding claims, wherein the preferred
encapsulated bacteria is selected from Lactobacillus rhamnosus, Lactobacillus
fermentum, Lactobacillus salivarious, Bacillus species {Bacillus subtilis),
Streptococcus species (Streptococcus thermophilus, Streptococcus salivarius),

Bifidobacteria {Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium
infantis) and Lactococci (Lactococcus lactis).

ABSTRACT

The present invention provides a process for the preparation of probiotic coated fennel
seeds, said process comprising the steps of:
i) Encapsulation of bacteria;
ii) Preparing primary coating comprising steps of:
a) coating fennel seeds by gradational addition of sugar syrup;
b) incubating the said coated fennel seeds at a temperature of 45-55°C
for 1-3 hrs
iii) preparing secondary coating comprising steps of:
c) Coating pre-coated fennel seeds with gradual addition of sugar syrup
at a temperature of 55-65°C for 1-3 hours, wherein said sugar syrup is
supplemented with encapsulated the bacteria,
d) Drying said coated fennel seeds at a temperature of 45-55°C for 2-3
hour.