FORM 2

THE PATENTS ACT,1970

(39 of 1970)

&

The Patents Rules, 2003

COMPLETE SPECIFICATION

(See section10; rule 13)

1. Title of the Invention–

“DEVELOPMENT OF BIOACTIVE FOOD PACKAGING

MATERIAL UTILIZING IMMOBILIZED BACTERIOCIN
BACICLAUSII-9 AND BANANA STEM WASTE”
2. Applicant(s)

NAME NATIONALITY ADDRESS

DR. YATI A 201 Ram kunj III app, Nanabazar,
HEMANTKUMAR INDIAN Vallabh Vidyanagar, Gujarat, India,
VAIDYA 388120
DR.
A 201 Ram kunj III app, Nanabazar,
DIVYESHKUMAR
INDIAN Vallabh Vidyanagar, Gujarat, India,
PRAMODRAI
388120
DAVE
Radha Swami Bungalows, Near
DR. SUNIL
Sonpriya Apartment, B/h D. Z. Patel
HRISHIKESH INDIAN
School, Anand, Gujarat, India,
CHAKI
388001
3. PREAMBLE TO THE DESCRIPTION
Complete Specification
The following specification particularly describes the invention and the manner in
which it is to be performed.

Signature Not Verified

Digitally Signed.
Name: Dadi Mohsin Jamilahmed
Date: 08-Sep-2023 22:35:54
Reason: Patent Efiling
Location: DELHI
 FIELD OF INVENTION:
The present invention relates to the field of the development of bioactive food
packaging materials. The present invention relates to the utilization of
immobilized Bacteriocin Baciclusii-9 and banana stem waste in the development
5 of bioactive food packaging materials. This present invention also relates to
enhance food packaging by providing a novel, eco-friendly solution with
improved food safety and extended shelf life.

BACKGROUND OF THE INVENTION

10 Wood pulping for paper production is a major contributor to deforestation and has
adverse effects on the environment. The need for more sustainable and
environmentally friendly alternatives in the paper industry has led to the
exploration of bioactive packaging materials. In this study, bioactive packaging
paper material was developed using purified bacteriocin and banana waste stem.
15 To obtain the bacteriocin, potent probiotic bacteria were isolated from human
breast milk. The isolated bacteria were screened for probiotic properties such as
pH and bile salt tolerance, antimicrobial activity, survival in simulated gastric and
upper intestine contents, auto-aggregation, co-aggregation properties, and cell
surface hydrophobicity. One strain of Bacillus clausii, exhibiting promising
20 probiotic properties, was identified at the molecular level.
The inhibitory effects of Bacillus clausii strains and a banana waste fiber film
containing Bacillus clausii-derived bacteriocin (Baciclusii-9) against foodborne
pathogens and spoilage microorganisms were evaluated through in vitro
antimicrobial screening. The results showed that both Bacillus clausii strains and
25 the banana waste fiber film exhibited inhibitory effects against pathogens such as
B. cereus, C. perfringens, P. aeruginosa, Listeria monocytogenes, and
Staphylococcus aureus, as well as fungi like F. oxysporum and C. gloeosporioides.
The bacteriocin derived from Bacillus clausii was then used as a functional agent
for the antimicrobial treatment of waste paper. The nanocomposite paper, formed
30 by incorporating the bioactive bacteriocin, was characterized for its
physicochemical properties. The minimum inhibitory concentration of the

2
 bacteriocin was determined to be 2 µg/mL. The thickness of the resulting
composting paper was measured to be 0.15-0.16 mm using a digital micrometer.
The synthesized bioactive paper exhibited a significant increase in tensile strength,
measuring 6.4 compared to the blank control, which measured 3.5. The water
5 absorption capacity of the bioactive paper was also slightly improved compared
to the blank paper. These findings suggest that functionalizing waste paper
through the incorporation of bioactive components, such as bacteriocins, can serve
as a sustainable waste management approach while enhancing the physical
properties of the resulting paper.
10 The present invention focused on the development of bioactive packaging paper
material using purified bacteriocin bacteriocin (Baciclusii-9) derived from
Bacillus clausii and banana waste stem. The incorporation of the bioactive
bacteriocin resulted in enhanced antimicrobial properties and improved tensile
strength of the composting paper. Such approaches contribute to sustainable waste
15 management and provide more environmentally friendly alternatives to traditional
wood-based paper production in the quest for a greener paper industry.
The problem addressed by this present invention is the need for bioactive food
packaging materials that can enhance food safety and extend shelf life.
Conventional packaging materials often lack antimicrobial properties, leading to
20 microbial contamination and food spoilage, resulting in foodborne illnesses and
economic losses.
This present invention proposes the utilization of immobilized Bacteriocin
Baciclausii-9 and banana stem waste to develop bioactive food packaging
materials. The immobilized bacteriocin offers controlled release of antimicrobial
25 compounds, effectively inhibiting microbial growth and improving food safety.
By incorporating banana stem waste as a raw material, sustainability is promoted,
reducing environmental impact. This innovative solution provides an eco-friendly
approach to enhance food packaging, offering improved food safety and extended
shelf life, thereby mitigating the problems caused by microbial contamination and
30 food spoilage.

3
 DESCRIPTION OF THE RELATED ART
Patent no. AU2011312184A1 disclosed subject matter relates to antimicrobial
packaging materials (such as films) useful in the packaging of foodstuffs and other
products. The presently disclosed subject matter also relates to processes for the
5 production of such materials, and to the use of the materials in antimicrobial
applications.
Patent no. KR20160067375A disclosed invention relates to a producing method
of an anti-microbial film for packaging foods, having excellent food spoilage
preventing function, and to an anti-microbial film produced therefrom and, more
10 specifically, to a producing method of an anti-microbial film for packaging foods
adding at least one herb medicine extract selected from the group consisting of
grapefruit seed, ginger, and Phellodendron bark as an anti-microbial additive,
which can maintain the freshness of the foods for a long time and can inhibit
bacterial decomposition, and to an anti-microbial film produced thereby.
15 Patent no. EP2965623B1 disclosed invention relates to modified polymeric
packaging materials having antibacterial, ethylene scavenging and barrier
properties, and more particularly the invention pertains to polymeric packaging
materials containing halloysite nanotubes with multifunctional properties.
Patent no. US11566116B2 disclosed invention is directed to methods for
20 preparing biologically active polymers via reactive extrusion, as well as the
resulting active resin and active packaging materials formed using such resin,
which possess a covalently attached active ligand that is therefore non-migratory
with respect to food stuffs stored while in contact with these packaging materials.
Patent no. WO2018106191A1 disclosed various embodiments disclosed herein
25 has antimicrobial / antifungal properties due to presence of a natural antimicrobial
agent as an additive in the packaging material. As such,
in food packaging applications for example, it may be possible to achieve a delay
or inhibition in the development of food spoilage microorganisms by inhibiting
undesirable micro-organisms from proliferating on food surfaces upon
30 contact of the packaging material with the food items. In so doing, food safety for
public health and an extended shelf-life of food products may be achieved.

4
 Advantageously, the packaging material according to embodiments disclosed
herein is an environmentally- friendly, green packaging material, and its
preparation involves a green fabrication step free from chemical additives and
toxic residues. The packaging material according to embodiments disclosed
5 herein may be particularly suitable as a food packaging material on
various food products such as confectionaries, seafood, red meat, and dairy
products, to name only a few.
Patent no. 202311037662 disclosed preparation method of packaging has low
initial cost as well as operational cost. The preparation method of packaging film
10 is presently not available for small and medium use in India due to nonawareness.
Spraying coating method has less uniformity of coating over fruit surface the
developed hydrogel film can be used on large scale in food industry for
enhancement of shelf life of food product. The swelling index of the developed
hydrogel film was such that only the dried food materials can be used for food
15 packaging. So, there should be more improvement of decreasing swelling index
instantly so that some products having low amount of water can also be packed
using hydrogel film. Guar gum was used for development of hydrogel film as key
material. The synthetic non-biodegradable plastics can be replaced by natural and
biodegradable film which can easily be decomposed and is eco-friendly. The
20 development of hydrogel film work was accomplished in three phases i.e., first
phase consisted of initiation of polymerization, second phase included cross
linking and the third phase involved the development of hydrogel film. The
invented packaging film aims to get maximum uniformity, biodegradability,
transparency, mechanical strength and decreased colour difference on
25 horticultural produce. The film can be recycled or reused after packaging of dry
horticultural produce or any other product. The developed packaging film was
aimed to increase capacity or strength of film for horticultural produce compare
to synthetic plastic films. The invented packaging film has an objective to provide
a low cost but the efficient film for low and medium weight produce producers.
30 Patent no. 202211011264 disclosed Disclosed herein a composition of Food
packaging material with use of only agricultural waste like corn cob or coconut

5
 husk or any other along with fruit peel waste in the ratio of 1:4 to 1:1 or 4;1 to 1:1
(specially use of citrus fruit peel waste like orange/lemon or pomela or any other);
Wherein adding of vinegar (100 to 300% w/w) for wetting of waste material and
making pulp in reactor; and Reactor temperature from 140 to 180°C with heating
5 from 2to 4 hrs; addition of aloe-vera (natural plant material or any other natural
plant adhesive or natural rubber) from 5 to 20% (w/w); wherein addition of
Eucalyptus oil (1 to 10 %) or any plant based essential oil having antimicrobial
property. Another embodiment, first 100gm agri-waste like corn cob, coconut
husk was air dried with about 20-40% moisture, grinded and dipped completely
10 in natural vinegar 100 to 300% (w/v g/ml) (by product of sugar cane process) for
24 to 48 hrs and then taken out mixed with dried fruit waste especially citrus fruit
peels like lemon/orange/chakotra in high temperature reactor/z blade mixer at 140
to 180°C for 2-4 hrs.

15 OBJECTS OF THE INVENTION

The main object of the present invention is related to develop a bioactive food
packaging materials.
Another object of present invention is to utilization of immobilized Bacteriocin
Baciclusii-9 and banana stem waste in the development of bioactive food
20 packaging materials
Yet another object of the present invention is to enhance food safety and extend
the shelf life of packaged food products by using bioactive food packaging
materials
Further object of the present invention is to enhance food packaging by providing
25 eco-friendly solution.

SUMMARY OF THE INVENTION

The present invention relates to the field of food packaging materials.
Specifically, it relates to the development of a bioactive food packaging
30 material that incorporates immobilized bacteriocin Baciclausii-9 and
banana stem waste, which provides enhanced antimicrobial properties to
6
 prolong the shelf life of packaged food products.
The preservation of food products during storage and transportation is a
critical concern in the food industry. Packaging materials play a significant
role in maintaining the quality and safety of food products by preventing
5 microbial growth, oxidation, moisture loss, and other factors that can lead
to spoilage and degradation. Conventionally, synthetic packaging materials,
such as plastic films, are widely used due to their low cost, versatility, and
ease of production. However, these materials have limited antimicrobial
properties, leading to shorter shelf lives and increased food waste.
10 In recent years, there has been a growing interest in developing bioactive
food packaging materials that can actively inhibit the growth of pathogenic
and spoilage-causing microorganisms. One promising approach is the
incorporation of natural antimicrobial agents, such as bacteriocins, into the
packaging material. Bacteriocins are proteinaceous antimicrobial
15 compounds produced by bacteria, which exhibit a broad spectrum of
activity against various foodborne pathogens and spoilage organisms.
Baciclausii-9 is a bacteriocin produced by a strain of Bacillus clausii, a
bacterium commonly found in the natural environment. Baciclausii-9 has
shown potent antimicrobial activity against a wide range of pathogens and
20 spoilage-causing microorganisms. However, to utilize its antimicrobial
properties effectively in food packaging, it is necessary to develop a suitable
delivery system that can immobilize Baciclausii-9 and release it gradually
over an extended period.
Additionally, there is a growing interest in utilizing agricultural waste
25 materials to develop sustainable and eco-friendly packaging materials.
Banana stems are a byproduct of banana cultivation and are often discarded
as waste. However, banana stems contain various bioactive compounds,
including phenolics and cellulose, which have been shown to possess
antimicrobial and antioxidant properties. By incorporating banana stem
7
 waste into the packaging material, not only can the waste be utilized
effectively, but also the overall antimicrobial activity of the material can be
enhanced.
The present invention addresses the need for an improved bioactive food
5 packaging material with enhanced antimicrobial properties. The invention
utilizes immobilized bacteriocin Baciclausii-9 and banana stem waste to
develop a novel packaging material that can actively inhibit the growth of
pathogenic and spoilage-causing microorganisms. The immobilization of
Baciclausii-9 ensures controlled release, prolonging its antimicrobial
10 activity over time. The incorporation of banana stem waste enhances the
overall antimicrobial properties of the packaging material while promoting
sustainability and waste utilization.
In conclusion, by utilizing the developed bioactive food packaging material,
food products can be preserved for a longer duration, reducing spoilage, and
15 extending their shelf life. This invention has the potential to revolutionize
the food packaging industry by providing an eco-friendly and effective
solution to enhance food safety and quality.
BRIEF DESCRIPTION OF DRAWINGS
It is to be noted, however, that the appended drawings illustrate only typical
20 embodiments of this invention and are therefore not to be considered for limiting
of its scope, for the invention may admit to other equally effective embodiments.
Figure 1 shows SDS-polyacrylamide gel electrophoresis of purified bacteriocin
from Bacillus Clausii: Bacteriocin was analyzed by SDS-PAGE on 12%
separating gel with a 5% stacking gel stained with Coomassie brilliant blue R-250
25 (Himedia). Lane 1: Ultra-low molecular weight protein marker. Lane 2: Column
purified bacteriocin
Figure 2 shows Antibacterial spectrum of bacteriocin produced by Bacillus clausii
at various purification stage against indicator microbes [A] B. cereus [B]
Staphylococcus aureus [C] C. perfringens [D] Pseudomonas aeruginosa [E]

8
 Listeria monocytogenes [F] Aspergillus niger [G] Colletotrichum gloeosporioides
Figure 3 shows UV-Visible spectroscopy of bioactive paper
Figure 4 shows bioactive food packaging material

5 DETAILED DESCRIPTION OF INVENTION

The present invention discloses to develop a bioactive food packaging materials
by the utilization of immobilized Bacteriocin Baciclusii-9 and banana stem waste
to enhance food safety and extend the shelf life of packaged food products by
providing eco-friendly solution.
10
Purification and identification of bacteriocin
The bacteriocin production process involved the use of Cell-free neutralize
supernatant (CFNS). The CFNS (crude bacteriocin) was first mixed with 40%
ammonium sulfate for protein precipitation and then stored at 4°C. The
15 precipitated proteins were collected by centrifugation at 10,000×g for 30 minutes
at 4°C. The resulting pellet was dissolved in 0.1M phosphate buffer with a pH of
7.0 and then dialyzed against the same buffer at 4°C overnight using a 10kD cutoff
membrane.
The partially purified protein was subjected to cation exchange chromatography
20 using an SP-Sepharose Fastflow column equilibrated with 50mM phosphate
buffer at pH 7.0. The absorbed proteins were eluted using a linear salt gradient (0
to 1M NaCl) at a flow rate of 2ml/min. The absorbance of protein fractions was
measured at 280nm, and fractions showing high bacteriocin activity were
collected and concentrated for further analysis.
25 The molecular weight of the purified bacteriocin was determined using 12%
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel
was stained with Commassie Brilliant Blue R-250 and destained with a mixture
of water, methanol, and acetic acid. Additionally, the purified fraction of the
protein was identified using Liquid Chromatography-Mass Spectrometry (LC-
30 MS/MS) for more detailed analysis and characterization.
The bacteriocin extraction and purification process utilized the cell-free

9
 neutralized supernatant of B. clausii. The purified bacteriocin demonstrated
significantly higher purity compared to the crude extract (Figure 1). The purified
bacteriocin displayed a broad spectrum of inhibitory activity against both gram-
positive and gram-negative pathogenic indicator strains after 24 hours of
5 incubation under suitable conditions (Figure 2).
In a quantitative analysis, the activity units of the bacteriocin were evaluated, and
a value of 4566 AU/mL was observed for all the tested indicator microbes. The
bacteriocin purity was found to be approximately 39.13 times higher than that of
the crude extract. Furthermore, the purified bacteriocin exhibited a specific
10 activity of 2.3 × 104 AU/mg against the indicator microbes (Figure 1). The
purified fraction of the bacteriocin contained a concentration of 1.2 mg/mL of
protein.
The results from SDS-PAGE analysis (Figure 1) revealed a single purified band
corresponding to the bacteriocin, with an apparent molecular weight ranging from
15 10-14 kDa. Additionally, the molecular weight of the purified bacteriocin was
further confirmed using Liquid Chromatography-Mass Spectrometry (LC-MS),
which indicated a molecular weight of 13.27 kDa.

Peptide sequence from Mass Spectra:

20 IIGLDALRIPAVQEAIKLVSVSLPRKSVKKTELSNSAHPYWGSQM
ELYIKIVDGVNQIDKDYGSDVCVVEGDISKKSVKALEEAKIADIL
KRLISVVPSLDTGVCDQQTRDVIATAIK

Synthesis of bioactive paper

25 Step 1: Harvesting and Chopping
Banana stems are obtained after harvesting the fruit and chopped into
intermediate-sized pieces, ranging from 1.5 inches to 2 inches.

Step 2: Sun Drying

30 The chopped banana stem pieces are sun-dried until the moisture content reaches
11% to 15%.
10
 Step 3: Chemical Retting
The sun-dried stem chippings are subjected to chemical retting using a 15% NaOH
solution. The concentration of the alkaline solution can vary depending on the
desired qualities of the pulp and fibers.
5
Step 4: Cooling
After cooking, the fibers are cooled for 20 to 30 minutes.

Step 5: Peroxide Treatment

10 The cooled sludge is treated with 40 ml of peroxide to remove color impurities
and odors. Continuous agitation of the slurry during the 20-minute treatment
enhances the effectiveness of peroxide.

Step 6: Neutralization
15 After peroxide treatment, the slurry is neutralized using 4-5 ml of 0.1N HCl.

Step 7: Dilution and Addition of Baciclusii-9 Bacteriocin

The treated pulp is diluted to form a slurry with a concentration adjusted to utilize
minimum water. Baciclusii-9 bacteriocin is then added to the slurry.
20
Step 8: Paper Formation
The diluted slurry with added bacteriocin is poured onto a wire woven screen of
70-80 mesh. A deckle is used to hold the slurry in place until the liquid passes
through the screen.
25
Step 9: Drying
Mechanical pressing removes residual water from the wet paper sheet, which is
then partially dried on the screen until it becomes stiff enough to be removed. The
removed paper sheet is subjected to final drying.
30
The process described above provides a sustainable and efficient method for

11
 producing high-quality Kraft paper from banana stem fibers. By incorporating
peroxide treatment and bacteriocin addition, the resulting paper exhibits improved
color and odor properties, making it suitable for a wide range of applications.
Additionally, the eco-friendly nature of the process contributes to environmental
5 conservation efforts.

Novel features of the present invention

Utilization of Immobilized Bacteriocin Baciclusii-9: The immobilization of
Bacteriocin Baciclusii-9 allows for its incorporation into the food packaging
10 material, enabling controlled release of antimicrobial compounds. This feature
enhances the packaging's ability to inhibit the growth of harmful microorganisms
and improve food safety.
Purification and Peptide Sequence Identification: The purification of Bacteriocin
Baciclusii-9 using the sp sapharose fast flow column ensures its high quality and
15 potency. Additionally, the identification of the peptide sequence using LC-
MS/MS techniques contributes to a better understanding of the antimicrobial
properties and aids in future applications.
Sustainable Raw Material: The use of banana stem waste as a raw material for the
packaging material aligns with sustainable practices. By repurposing agricultural
20 waste, the invention contributes to reducing environmental impact and promotes
a circular economy.
Enhanced Food Safety and Extended Shelf Life: The incorporation of
immobilized Bacteriocin Baciclusii-9 and the antimicrobial properties of the
packaging material contribute to improved food safety. This innovation helps to
25 inhibit the growth of pathogens and spoilage microorganisms, thereby extending
the shelf life of packaged food products.
Novel, Eco-friendly Solution: The combination of immobilized Bacteriocin
Bacillusii-9 and banana stem waste results in a unique, eco-friendly solution for
bioactive food packaging. This inventive approach offers an alternative to
30 conventional packaging materials, providing a sustainable and efficient option for
enhancing food quality and safety.

12
 WE CLAIM:

1. A sustainable bioactive paper production process utilizing banana stem

fibers, comprising the steps of:
5 a. Harvesting and chopping banana stems into intermediate-sized
pieces;
b. Sun-drying the chopped banana stem pieces to achieve a moisture
content of 11% to 15%;
c. Subjecting the sun-dried stem chippings to chemical retting using a
10 15% NaOH solution;
d. Cooling the retted fibers;
e. Treating the cooled sludge with peroxide to remove color impurities
and odors;
f. Neutralizing the peroxide-treated slurry using 0.1N HCl;
15 g. Diluting the neutralized slurry and adding Baciclusii-9 bacteriocin;
h. Pouring the diluted slurry with added bacteriocin onto a wire woven
screen for paper formation;
i. Drying the wet paper sheet through mechanical pressing and final
drying to produce bioactive paper.
20 2. The sustainable bioactive paper production process as claimed in claim 1,
wherein the resulting bioactive paper exhibits improved color and odor
properties compared to conventional paper made from banana stem fibers.
3. The sustainable bioactive paper production process as claimed in claim 1,
wherein the bioactive paper displays a broad spectrum of inhibitory
25 activity against both gram-positive and gram-negative pathogenic
indicator strains.
4. The sustainable bioactive paper production process as claimed in claim 1,
wherein the bacteriocin, Baciclusii-9, is purified through cation exchange
chromatography and exhibits a molecular weight of 13.27 kDa along with
13
 peptide sequence
IIGLDALRIPAVQEAIKLVSVSLPRKSVKKTELSNSAHPYWGSQM
ELYIKIVDGVNQIDKDYGSDVCVVEGDISKKSVKALEEAKIADIL
KRLISVVPSLDTGVCDQQTRDVIATAIK
5

10

15

20

FOR DR. YATI HEMANTKUMAR VAIDYA

DR. DIVYESHKUMAR PRAMODRAI DAVE AND
DR. SUNIL HRISHIKESH CHAKI

25
Dadi Mohsin Jamilahmed
Registered Patent Agent (IN/PA-4315)
Agent for the Applicant
Dated: 07th September, 2023
14
 ABSTRACT
DEVELOPMENT OF BIOACTIVE FOOD PACKAGING MATERIAL
UTILIZING IMMOBILIZED BACTERIOCIN BACICLAUSII-9 AND
BANANA STEM WASTE

The present invention relates to develop bioactive packaging paper material by

incorporating purified bacteriocin and banana waste stem. Probiotic bacteria were
isolated from Human breast milk and screened for probiotic properties. Bacillus
clausii strains with desirable probiotic properties were identified and evaluated for
their inhibitory effects against foodborne pathogens and spoilage microorganisms.
A banana waste fiber film containing the antimicrobial bacteriocin Baciclusii-9
was also assessed for its inhibitory effects. Both the strains and the banana waste
fiber film showed inhibitory effects against various pathogens and fungi. The
bacteriocin was utilized as a functional agent for antimicrobial properties in
wastepaper. The physicochemical characterization of the nanocomposite paper
revealed a thickness of 0.15-0.16 mm and a significant increase in tensile strength
compared to the blank control. The water absorption capacity of the composting
paper was also determined. The results suggest that incorporating bioactive silver
nanoparticles into wastepaper could serve as a sustainable waste management
approach.
FOR DR. YATI HEMANTKUMAR VAIDYA
DR. DIVYESHKUMAR PRAMODRAI DAVE AND
DR. SUNIL HRISHIKESH CHAKI

Dadi Mohsin Jamilahmed

Registered Patent Agent (IN/PA-4315)
Agent for the Applicant
Dated: 07th September, 2023

15