1.
Name of Institute:- CIFE Mumbai
Project 1:Title:- Development of Nano-Sized Delivery system for Nutraceuticals in
Aquafeed
Name of the PI:- Dr. Subodh Gupta
Main objectives:
1. Major objective of this project is to design nano-sized delivery system intended
for the optimum utilization of nutraceuticals that
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Protect the associated enzyme from degradation in the gastro-intestinal
fluid.
Enhance the rate of absorption in the gastro-intestinal tract.
Enhance bioavailability and increase nutrient dynamics.
Reducing nutrients excretion into the environment and decrease
environmental pollution.
Ultimately enhancing growth, health status and survival rate
2. To check the efficiency of these nano-sized delivery system in fish model.
3. To design dispersible feed pellet containing these formulated nanoparticles for
efficient and cost effective feed for aquatic animal.
Report:
Nanotechnology is being increasing sought to improve use of nutrients /nutraceuticals
for disease prevention and enhance performance. Advances in the understanding of
gastrointestinal tract physiology have enabled the manipulation of digestibility and
absorption of food constituents, chemicals and biomolecules to meet individual needs
and goals in farm animal production. Exogenous nutraceuticals supplementation is an
important area of research in humans and animals.
Development of Nano-Sized Delivery system for Nutraceuticals
a) Chitosan encapsulated trypsin nanoparticle
People with exocrine pancreatic insufficiency and cystic fibrosis frequently require
supplemental pancreatic enzymes (which include proteolytic enzymes, lipases, and
amylases). In addition, some people suffering from celiac disease or Crohn’s disease
and perhaps from indigestion may be deficient in pancreatic enzymes or required more
proteotytic enzymes to optimum digestion of dietary supplemented protein because
protein is the most costly feed ingredients. Exogenous enzymes in animal feed are
used for compelling reasons: shortage of preferred animal protein sources (fish meal),
coupled with greater use of plant protein ingredients as well as the profitability in the
fish, poultry and pig sectors, which are compete for feed resources among themselves
and also with humans.
The innovative idea for nano-encapsulation of trypsin with chitosan is that as pancreas
gland secretes trypsin as trypsinogen or in inactive form (zymogen) and will activate
in gastrointestinal tract by proteolytic cleaving that converts to active trypsin to check
autolysis and protect the delicate epithelial mucosa of gastrointestinal tract and usually
in animal feed, we add active form. This nanoformualated trypsin proved that nanoencapsulation of trypsin not only protects the delicate epithelial mucosa but also
improve the protein digestion in compare to bared form trypsin.
Characterization of chitosan encapsulated trypsin nanoparticle (CE-Try-NPs)
Chitosan nanoparticles (CNPs) were prepared based on the modified ionic gelation
method (Calvo et al., 1997). These synthesized CNPs were used for the production of
nanoencapsulated enzyme for oral delivery of exogenous enzymes in Labeo rohita
fingerlings. Two different level of trypsin and 0.4% of chitosan were taken for
nanoparticle preparation i.e., 0.4% chitosan with 0.01% trypsin and 0.02% (refereed
as 0.01% CE-Try-NPs and 0.02% CE-Try-NPs respectively) presented in figures 1-3.
Fig1: Particle size distribution of 0.4% CNPs (n=3)
Fig2: Particle size distribution of 0.01% CE-Try-NPs (n=3)
Fig3: Particle size distribution of 0.02% CE-Try-NPs (n=3)
Zeta potential measurements
The zeta potential of CNPs and CE-Try-NPs was measured. The measurement of zeta
potential predicted about the stability of nanoparticle and presented in figure 4.
Zeta potential (mV)
Zeta potential (mV)
Chitosan
0.01 Cnet
0.02 Cnet
0.4% CNPs
0.01% CE-Try-NPs
0.02%CE-Try-NPs
Fig Fig 4: Zeta potential value of CNPs and CE-Try-NPs
AFM results
AFM analysis of the chitosan nanoencapsulated trypsin revealed that
the nanoparticles exhibited spherical shape with a homogenous distribution (Figure 5
and 6).
Fig 5: Three-dimensional image of 0.01%CE-Try-NPs
Fig 6: Three-dimensional image of 0.02% CE-Try-NPs
Histological study of intestine indicates safety of nanoencapsulated trypsin
over its bare form
Intestine histology of Labeo rohita fingerlings fed the control diet showed intact
architecture (Fig 7A), and histology of 0.4%chitosan NPs-fed fish did not show much
difference from control (Fig 7B). Intestinal tissues of fish fed with the 0.02% bare
trypsinsupplemented diet showed broadened villi, marked foamy cells with lipid
vacuoles, atrophied submucosal layer and muscularis(Fig 7C-1 and 7C-2). In general,
villi were healthier in appearance and had improved morphological features after being
fed chitosannanoencapsulated trypsin compared to bare trypsin. The villi were longer
in fish fed with 0.01% chitosan nanoencapsulated trypsin (Fig 7D) than 0.02% chitosan
nanoencapsulated trypsin, which slightly resembled the control group (Fig 7E).
Fig 7:Intestine histology of fish fed control, 0.4% chitosan nanoparticles, 0.02%
bare trypsin and and 0.01% or 0.02% nanoencapsulated trypsin containing diets
(H&E, 40X). In control group (A), intestinal mucosa is lined by regularly-packed
villi withcontinuous basement membrane. In 0.4% chitosan nanoparticles fed
fish (B), mildly swollen apical surface of villi (1) are noticed. In 0.02% bare
trypsin (C-1 and C2), broadened villi, marked foamy cells with lipid vacuoles (2),
atrophied submucosal layer and muscularis (3) were evident. In 0.01% trypsin
encapsulated in 0.4% chitosan nanoparticles (D), longer villi (4) with healthy
apical surface (5) and improved morphological features besides continuous
basement membrane are evident. Crypt depth was less in treated groups (6).
Distinct mucin producing goblet cells (7) distributed along the villi in
nanoencapsulated trypsin groups indicates better gastro-intestinal health. Villi
in 0.02% trypsin encapsulated in 0.4% chitosan nanoparticles (E) resembled
those in control group (A). Effectiveness and safety of dietary
nanoencapsulated trypsin at half the dose rate (0.01%) (D) of bare trypsin
(0.02%) (C-1 and C-2) is evident from healthier villi with more height and
absorptive surface.doi:10.1371/journal.pone.0074743.g007
b) RNA-loaded chitosan nanoparticles
Exogenous nucleotide supplementation during times of rapid growth and stress is
preferred because de novo synthesis is insufficient and energetically a costly process.
To overcome inefficient utilization of dietary nucleotides dueto intestinal cell repulsion
and dependency on pH, an efficient controlled delivery system based on chitosan
nanoparticles (NPs) was developed.
Characterization of Chitosan and RNA-Loaded Chitosan NPs
Comparison of the physical and chemical characteristics of the synthesized RNAloaded chitosan NPs (Table 1) indicated that as the proportion of chitosan increased,
the size and so the zeta potential of the nanoparticles were increased significantly (P
< 0.01). The optimum entrapment efficiency of RNA-loaded chitosan nanoparticles
was found where chitosan to RNA ratio was 2:1; it was decreased where the ratio was
3:1. Particle size distribution of the different ratios of the chitosan and RNA is shown
in Fig. (8A-C). Two dimensional (Fig. 8B-1) and three dimensional (Fig. 8B-2) images
by atomic force microscopy of the RNA-loaded chitosan NPs revealed that the
nanoparticles exhibited a spherical shape with a homogenous distribution.
Table 1. Physico-chemical characteristics of chitosan
nanoencapsulated RNA varying in chitosan to RNA ratio
Chitosan:RNA Ratio¶
and
chitosan
Particle size Zeta potential Entrapment
(nm)
(mV)
efficiency %
Chitosan: RNA (1:0) (Chitosan 126.4±1.46
34.8±0.53
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blank)
Chitosan: RNA (1:1)
250.1a±1.58
27.1a±0.62
61b±0.97
Chitosan: RNA (2:1)
287.1b±1.49
34.5b±0.49
73.5c±0.85
Chitosan: RNA (3:1)
326.8c±1.21
37.0c±0.46
56.5a±1.16
¶Chitosan: RNA ratio was varied by using different chitosan concentrations (0.4, 0.8
and 1.2 percent) but keeping RNA concentration same i.e. 0.4 percent for all the
combinations a, b, c Means of a physico-chemical character of chitosan: RNA
nanoparticles bearing different superscript letters differ significantly (P < 0.01). Values
represent means ± standard error of triplicate observations.
Fig 8:Particle size distribution of nanoparticles and atomic force microscope
(AFM) images. RNA-loaded chitosan NPs were prepared with varying ratios of
chitosan to RNA such as 1:1 (A), 2:1 (B) and 3:1 (C). As NPs prepared with a
chitosan to RNA ratio of 2:1 were used for further study, two-dimensional (B-1)
and three-dimensional (B-2) images of these are shown.
Performance, Body Composition and Organo-Somatic Indices
The data on growth and performance (Fig. 9) indicated that feeding with plain chitosan
NPs had no effect, but feedingbare RNA increased (specific) growth (rate) (SGR) and
improved feed conversion (ratio) (FCR) as well as proteinefficiency (ratio) (PER).
Nano-sizing by loading of RNA onto chitosan NPs further potentiated its nutritional
benefits;which was evinced by significantly (P < 0.01) improved SGR (without effect
on FCR and PER) at half the dose ofbare RNA. When the dose rate of RNA was the
same, the productive efficiency in terms of SGR, FCR and PER withRNA-loaded
chitosan NPs was significantly (P < 0.01) higher than bare RNA. The whole body mean
per cent (n =3) protein (15.48±0.11 to 16.24±0.09), lipid (2.89±0.45 to 3.67±0.50) and
ash (1.78±0.37 to 2.24±0.24) content was notsignificantly different among different
groups. Feeding plain chitosan NPs had no effect on hepatosomatic index (HSI) and
viscero-somatic index (VSI) (Fig. 9). There was no effect from RNA in bare form, but
feeding thenano-sized form of RNA both at half the dose rate and at the same dose
rate of bare RNA resulted in a significant increase (P < 0.01) in HSI. The RNA in bare
form increased (P < 0.01), and nano-sizing had no further effect on GSI.
Fig 9:Performance and organo-somatic indices of fish fed chitosan
nanoparticles (NPs), bare RNA and RNA-loaded chitosan NPs for sixty days.
Performance (A) and organo-somatic indices (B). Bars bearing different
superscript letters differ significantly (P < 0.01). Values represent means ±
standard error of triplicate observations. Abbreviations: Chitosan NPs (CtNPs),
RNA-loaded chitosan NPs (RNA-CtNPs).
Project 2:Title:- Fish gelatain based nanocomposite films for food packaging
Name of the PI:- Dr. B.B. Nayak.
Main Objectives: To develop natural polymer based biodegradable films and study their mechanical
properties.
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To incorporate natural and synthetic nanomaterials improve the desirable qualities of the
films.
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To study the protective effect of natural preservatives added in the films
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To evaluate the efficiency of the developed films for extension of shelf life of fish
products
Reports:Not Submitted