exploitation of fermented shrimp-shells hydrolysate as...

Indian Journal of Experimental Biology

Vol.51, November 2013, pp. 924-934

Exploitation of fermented shrimp-shells hydrolysate as functional food:

Assessment of antioxidant, hypocholesterolemic and prebiotic activities

Suman Kumar Halder, Atanu Adak, Chiranjit Maity, Arijit Jana, Arpan Das, Tanmay Paul, Kuntal Ghosh,

Pradeep Kumar Das Mohapatra, Bikas Ranjan Pati* & Keshab Chandra Mondal

Department of Microbiology, Vidyasagar University, Midnapore 721 102, India

Received 9 December 2012; revised 16 February 2013

In the present study the bioactivities of chitooligosaccharides of fermented shrimp-shell hydrolysate (SSH) in respect to

hypocholesterolemic, antioxidant and prebiotic activity were tested in male albino rat. Rats were treated with four different

diets, viz., (i) cholesterol-rich (5%) basal diet (ChB), (ii) ChB+10% chitin, (iii) ChB+10% SSH and (iv) control group

(without cholesterol). After 4 weeks of treatment, body mass index, liver weight, serum total cholesterol and

LDL-cholesterol in groups (ii) and (iii) were decreased significantly than group (i). SSH supplementation significantly

resists oxidative stress by reducing the thiobarbituric acid reactive substances and by increasing catalase, superoxide

dismutase and free radical scavenging activity. The colonization of Lactobacillus and Bifidobacterium population in small

and large intestine were more in group (iii) than other groups. Reduction of Clostridium perfringens population and

non-significant changes of E. coli was also noted in SSH supplement group. Histological study revealed that the villus

height and villus:crypt of the small intestine were increased significantly in SSH supplemented group (iii) without any

diarrheal symptoms. The results demonstrated that the shrimp-shells hydrolysate has hypocholesterolemic effect, can resist

lipid peroxidation and can influence the growth of health beneficial microbes, hence can be used as functional food for

hypercholesterolemic patients.

Keywords: Aeromonas hydrophila, Antioxidant, Functional food, Hypocholesterolemic, Oxidative stress, Shrimp-shells

hydrolysate

Functional foods are natural and processed foods

contain biologically-active compounds which showed

significant health beneficial effect1. In this context,

oligosaccharides can be considered as effective

functional food ingredients considering its health

promoting effect as like as other documented

functional food1,2

. Traditionally, oligosaccharides are

defined as polymers of monosaccharides with degrees

of polymerization (DP) between 2-10 (3-10 according

to the IUB-IUPAC nomenclature) but DP up to

20-30 are often considered. The functional

oligosaccharides like galacto-oligosaccharide manno-

oligosaccharide, fructo-oligasaccharide are

non-cariogenic, non-digestible (by consumers) and

low caloric compounds3. Their consumption

influences the growth of health beneficial microbes

particularly Bifidobacteria and Lactobacilli,

alterations in the gastrointestinal tract architecture and

function, changes in adapting to enteric microbiota

and immune responses4,5

and thus, an important

source in the prevention, management and treatment

of chronic diseases of the modern era2.

Oligosaccharides have currently two origins: they can

be synthesized by chemical glycosylation and de novo

using glycosidase and glycosyl transferase activities,

or they can derive from chemical, physical or

biological degradation of polysaccharides. Due to

numerous incompatibilities and evil effects of the

chemical hydrolysis, biological degradation becomes

more viable and well-liked.

Hypercholesterolemia is phenomenon of the

elevated levels of cholesterol in the blood. As

cholesterol is insoluble in water, it is transported in

the blood plasma within protein particles called

lipoproteins6. All the lipoproteins carry cholesterol

and among them high density lipoprotein-cholesterol

(HDL-C) are defensive6. On the contrary, elevated

levels of low density lipoprotein-cholesterol (LDL-C)

and triglycerides (TG) are associated with an

increased risk of dyslipidemia like atherosclerosis,

coronary heart disease and cardiovascular diseases7.

Fifty percent of mortality in developed countries and

——————

*Correspondent author

Telephone: 03222-276554/555 (Ext. 477)

Fax: 03222-275329

E-mail: [email protected]

HALDER et al.: SHRIMP SHELL HYDROLYSATE AS FUNCTIONAL FOOD

925

25 % deaths in the developing world are due to the

diseases related to atherosclerosis with dyslipidemias

being its root cause.

On the other hand, oxygen-derived free radicals or

reactive oxygen species (ROS) have been implicated

in the pathogenesis of a wide spectrum of diseases as

well as in the aging process8,9

. In addition to playing a

role in direct tissue damage, their generation may also

amplify the body’s general inflammatory response

and promote further cell injury8. In general, the

mammalian cell has adequate antioxidant reserves

including enzymes like catalase (CAT) and

superoxide dismutase (SOD) to cope with ROS

production under normal physiological conditions.

When the equilibrium between free radicals

(oxidants) and antioxidant defense systems is

imbalanced in favour of oxidants, the condition

causes what is known as oxidative stress9,10

. There are

many reports indicating the alteration of antioxidant

parameters during hypercholesterolemia induced

oxidative stress11

.

In India, about 300 seafood processing industries

annually produce about 1 lakh ton of shrimp-shells as

waste, which is a rich source of chitin12

.

Chitooligosaccharide (COS) can be efficiently derived

by valorizing these chitinous biowaste through

microbial fermentation by cost effective means. More

recently, COS has been shown to have immune-

enhancing characteristics13

and protection against

pathogenic infections14

. Extensive works already have

been carried out on galacto-oligosaccharide, manno-

oligosaccharide, fructo-oligasaccharide to ascertain

their beneficial bioactivities; however study on the

bioactivity of COS with respect to their degree of

polymerization is still inadequate.

In this perspective, the present study has been

conducted for production of COS by fermenting

chitinous shrimp shells as cost-effective substrate.

The health beneficial effects of COS in respect to

cholesterol lowering, growth promoting, antioxidant

and prebiotic activities have been examined in male

albino rat model.

Materials and Methods The study was conducted with ethical approval

from the Vidyasagar University Ethics Committee.

Animal were reared and fed according to suggested

rules of the Committee.

Preparation of shrimp shell hydrolysate (SSH)—

Shrimp (Penaeus sp.) shells were obtained from sea

food processing center of Shankarpur, West Bengal,

India and transported into the laboratory in frozen

condition. After thorough washing with tap water, the

shells were dried at 60 ± 2 ºC and ground to flakes by

electric blender of final particle size ~2mm. Solid

state fermentation of the shrimp shells was carried out

by chitinolytic bacteria Aeromonas hydrophila SBK1

(GenBank Accession no. HM802878.1) for 3 days at

37 ºC which was isolated by Halder et al15

. The

Shrimp shell hydrolysate (SSH) was then extracted by

water and purified by membrane filtration (Amicon

5 kDa cut-off) in single step. The filtrate was freeze

dried to powder and used as feed supplement.

MALDI-ToF analysis of SSH—The molecular mass

of the COS present in SSH was determined by

Voyager DE ProTM mass spectrometer equipped

with 337 nm N2 laser (Applied Biosystem, USA). SSH

solution (2 µL) was mixed with 24 µL of DHB

(2,5-dihydroxy benzoic acid) 10 mg/mL which was

used as matrix. Then, 1.0 µL sample was spotted onto

the 100 well stainless steel MALDI plate and allowed

to air dried prior to the MALDI analysis. The degree of

polymerization (dp) of COS was calculated from their

peak intensities in the MALDI-ToF mass spectrum.

TLC analysis of SSH and antioxidant—The

antioxidant materials of SSH were analyzed on pre-

coated silica gel thin layer chromatography (TLC)

plates (0.25 mm) (Merck, Germany) using 5:4:3

(v/v/v) n-butanol/methanol/16% aqueous ammonia as

the mobile phase16

. After developing the TLC plates,

the compounds were visualized by spraying with

ethanol containing 0.5% (w/v) ninhydrin (ninhydrin

reagent) and DPPH solution (0.75 mM in methanol),

separately, followed by heating.

Cytotoxicity assay—For this study Vero cells were

seeded into 24-well culture plates (Falcon) at a

density of 105 cells/well and incubated until reaching

at least 95% confluency. Different concentrations of

SSH were added to each culture wells at a final

volume of 100 µL using DMSO (0.1%) as a negative

control. After incubation at 37 °C with 5% CO2 for

2 days, MTT reagent (10 µL) was added to each well.

After 4 h of incubation at 37 °C, the formazan was

solubilized by adding diluted HCl (0.04 N) in

isopropanol, and the absorbance was read at 570 nm

with a reference wavelength of 690 nm by an ELISA

microplate reader. Data were calculated as the

percentage of cell viability using the formula:

[(sample absorbance - cell free sample blank)/ mean

media control absorbance)]/100%.

INDIAN J EXP BIOL, NOVEMBER 2013

926

Animal experiment—Healthy male albino rat (Rattus

albus) having the average body weight of

120 ± 5 g were used as model animal in the present

study. They were housed in metal made cages

(34 × 28 × 19 cm3). The experimental animals were

maintained with proper care with a photoperiod

of D:L (12:12) and without interrupting their

normal activity in a temperature-controlled nursery

(27 – 28 °C) during the entire 4 weeks experimental

period. Rats had access to feed and water ad libitum.

They were assigned to 4 treatments (n = 20), in a

randomized manner. The 4 treatments were (i)

cholesterol rich (5%) basal diet, (ii) cholesterol rich

(5%) basal diet supplemented with 10% chitin (60%)

deacetylated), (iii) cholesterol rich basal diet

supplemented with 10 % SSH, (iv) basal diet without

cholesterol (control group). The basal diet containing

carbohydrates (74.05%); proteins (10.38%); fibre

(2.20%); iron (56 ppm); calcium (400 ppm) and

sodium (500 ppm) was formulated to meet the

nutrient requirements suggested by Ethics Committee,

Vidyasagar University. All the ingredients were

premixed and boiled before serving.

General observation on physiological

parameters—The body weight and length (nose-to-

anus) and body weight were taken during

experimental period. Feed intake of the rats was also

evaluated at regular basis. The body mass index

(BMI) was determined using the formula:

Body mass index (BMI) =body weight (g)/length2

(cm2)

Blood and tissue sampling—After 4 week of

feeding, the rats were fasted overnight, and blood was

collected from the abdominal aorta under diethyl ether

anesthesia and kept in a covered sterile vial for

20 min. The clot was removed by centrifuging at

1000-2000 g for 10 min in a refrigerated centrifuge

(Remi R-24). Next to that, rats were deeply

anesthetized and liver, kidney, intestine were

dissected and immediately frozen and stored at -20 °C

until needed for analysis. Prior to analysis, liver,

kidney and intestine were homogenized in cold

phosphate buffer saline (pH 7.2) and supernatant was

used to analysis.

Determination of lipid profile—Total cholesterol,

HDL-cholesterol and LDL-cholesterol were measured

by using commercial kit method (Span Diagnostics

Ltd., India). Total lipid, triglycerides, cholesterol

from the tissue and feces were assayed according

to the methods of Carlson and Goldfard17

from the

total lipid extracted following the protocol of

Floch et al18

.

Antioxidant enzyme and lipid peroxidation

profiling—Catalase (CAT) activity was measured

according to the method of Aebi19

by following the

decrease in absorbance of H2O2 at 240 nm.

Superoxide dismutase (SOD) activity was measured

by the inhibition of pyrogallol autoxidation at 420 nm

according to the method of Marklund and Marklund20

.

Protein content was determined using the method of

Lowry et al.21

using bovine serum albumin as the

standard. One enzyme unit is defined as the amount of

enzyme that transformed 1 µmol substrate to product

per min under standard assay conditions. Lipid

peroxidation was measured as thiobarbituric acid

reactive substances (TBARS) by the thiobarbituric

acid colour reaction for malondialdehyde (MDA)

formation following the method of Beuge and Aust22

.

Bacteriological analysis—The quantities of

predominant cultivable indicator groups of large and

small intestinal bacteria were enumerated on the basis

of colony forming units (cfu) through dilution plating

technique. Selective media were used following the

standard protocol set out in the Hi-Media Manual

followed by Maity et al23

. Enumeration of E. coli and

Bifidobacterium spp. was carried out using selective

media such as MacConkey and Bifidobacterium agar

(Hi-Media), respectively. For selective cultivation and

enumeration of Lactobacillus spp. and Clostridium

perfringens, MRS agar and reduced perfringens

agar base (Hi-Media) were used respectively.

For anaerobic culture an anaerobic CO2 incubator

[Heal force Air jacket (HF 151 UV, 1unit)] was used.

The bacterial population was expressed as colony

forming unit (cfu). The cfu values were converted to

their logarithmic value and compared with the

corresponding experimental set. If the log value of

control cfu is higher than the log value of test

(any diet group) cfu, then growth direction index

(GDI) is designated as negative and the reverse event

is designated as GDI positive23

.

Diarrheal score—The incidence of diarrhea of rats

was observed and recorded 3 times per day during the

study. To assess the severity of diarrhea, feces from

each albino rat was scored by determining the

moisture content according to the method of Hart and

Dobb24

. Scores were 0, normal, firm feces; 1, possible

slight diarrhea; 2, definitely unformed, moderately

fluid feces; or 3, very watery and frothy diarrhea.

A cumulative diarrhea score per diet and day was then


927

calculated25

. The occurrence of diarrhea was defined

as maintaining fecal scores of 2 and 3 for

2 consecutive days.

Small intestinal morphology—Intestinal tissues

were fixed and paraffin sections (6 µm) were prepared

following conventional method. The tissue slices were

stained with hematoxylin and eosin. All analyses were

performed in a blinded fashion26

; villus height

and crypt depth were measured at 40X magnification

using Nikon Eclipse LV100POL light microscope

(Tokyo, Japan) with digital camera (Tokyo, Japan)

and Image was analyzed by Software Package NIS-

Element F3.0. A minimum of 15 well-oriented, intact

villi were selected to measure in triplicate for each rat.

Scanning electron microscopy (SEM)—After

collection of the part of small intestine it was rinsed

with cold normal saline, and cut into 2 mm × 2 mm

sections which were fixed in 2.5% glutaraldehyde and

10% osmium, dehydrated in sucrose solution

containing PBS. Simultaneously it was gold coated

and observed under scanning electron microscope.

The arrangement of villi of colon epithelial cells was

observed and special attention was paid to the

deformed and exfoliated villi and the intercellular

space between epithelia.

Statistical analysis—All experiments were performed

in triplicates and represented as mean ± SD. Statisticlal

analysis was performed in Sigma Plot 11 (USA).

Results

Analysis of shrimp shell hydrolysate—The

fermented shrimp shells hydrolysate (SSH) was

principally composed of chitooligosaccharides (COS)

upto degree of polymerization (DP) of 30 and showed

paramount DPPH radical scavenging activity. The

COS was identified through thin layer

chromatography as well as mass analysis from

MALDI-ToF spectrum (Fig. 1).

Cytotoxicity assay of COS—The dose dependent

MTT assay was used to evaluate the cytotoxic effect

of the SSH. The result revealed that the Vero cells

were alive and metabolically well active up to SSH

concentration 1000 µg/mL.

Growth performance—There was no significant

difference in the average daily feed intake among the

experimental groups. After 4 week of treatment, body

mass index (BMI) of the cholesterol rich diet group

(Group i) was significantly increased than the control

group (Group iv) (P<0.05) (Table 1). Conversely,

supplementation of chitin (Group ii) and SSH

(Group iii) leads to maintain the change in BMI close

to control group.

Serum lipids profile—The level of serum

triglycerides, total cholesterol (TC), LDL-cholesterol,

atherogenic index (TC/HDL-cholesterol) were

significantly lower (P<0.05) in animals fed on chitin

(Group ii) and SSH (Group iii) diets than the

cholesterol rich diet group SSH (Group i) (Table 1).

However, no significant change in HDL-cholesterol

was apparent among the dietary groups.

Liver weight and lipid content—The relative liver

weights of the chitin diet group (5.7 ± 0.3 g/100 g

body weight) and SSH diet group (5.8 ± 0.3 g/100 g

body weight) were significantly lower (P<0.05) than

the cholesterol rich diet group (6.7 ± 0.3 g/100 g

Fig. 1—Analysis of chitooligosaccharides (COS) of shrimp shell hydrolysate (SSH) through MALDI-ToF(A), the numerical numbers in

the spectrum indicates the degree of polymerization; thin layer chromatography with their free radical scavenging activity (B), M- COS

standard, C-COS of SSH, F-free radical scavenging activity by DPPH treatment of TLC plate.


928

body weight) and close to the control group

(5.5 ± 0.3 g/100 g BW) (Table 1).

The liver lipid profiles of the different dietary

groups are shown in Table 1. The results revealed that

supplementation of chitin and SSH down regulate and

minimize the total lipid, total cholesterol and

triglycerides contents of the liver significantly

(P<0.05) which were raised due to intake of high

level of dietary cholesterol (Group I). However, the

performance of chitin was superior over SSH in this

regard.

Plasma, liver and kidney lipid peroxidation and

antioxidant enzymes—Fig. 2 shows the lipid peroxide

profiles in the serum, liver and kidney of the rats of

different diet groups. Higher levels of lipid peroxide

values (by estimating MDA) were noticed in

cholesterol rich diet group in serum, liver and kidney.

The elevated MDA level was significantly lessened

by supplementing chitin and SSH (P<0.05). Though

the lipid peroxide value of chitin supplemented diet

group was less than SSH diet group, the difference

was insignificant. On the contrary, the CAT and SOD

activity in serum, kidney and liver of SSH treated

group were significantly higher than all the other

dietary groups (P<0.05).

Fecal lipid and diarrheal score— As shown in

Table 2, significantly higher level of fecal total lipid,

total cholesterol and triglycerides contents were found

in the feces of rat fed with chitin and SSH

supplemented diet (P<0.05) than cholesterol rich diet

group. On the contrary, diarrheal incidence due to

high cholesterol consumption (diarrheal score 13)

was significantly minimized (P<0.05) by SSH

(diarrheal score 3) than chitin supplementation

(diarrheal score 6).

Microbial analysis—Effects on different diets on

the predominant colonic microbiota are presented in

Fig. 3. It was evident that the cholesterol

supplementation significantly inhibited the growth of

Bifidobacterium (GDI -2.22 and -1.10) and

Lactobacillus (GDI -1.33 and -1.30) (P<0.05) in small

and large intestine respectively, whereas chitin

supplementation along with cholesterol rich diet

exhibited significant antibacterial property (P<0.05)

against the four bacterial community tested.

Interestingly, the supplementation of SSH selectively

propelled the growth and colonization of the

Lactobacillus (GDI 1.13 and 1.56) and

Bifidobacterium (GDI 1.21 and 1.39) groups and

reduced the count of C. perfringens (GDI -1.29 and -

1.18) in both small and large intestine respectively

(P<0.05). However, change in E. coli population due

to SSH supplementation was insignificant.

Small intestinal morphology—Histological study

revealed that there was a significant increase in

the villus:crypt ratio (2.8) in the rats (SSH

supplemented group) in comparison to the others

dietary groups (P<0.05) (Fig. 4). SEM analysis

revealed the enhanced colonization of colonic bacteria

in SSH treated group with regular epithelial

Table 1—Effect of different diet schedule on the growth performance and lipid profiles in male albino rats after 28 days of treatment

[Values are mean ±SD from 20 animals in each group of triplicate determinations]

Parameters Control basal diet

(without cholesterol)

Cholesterol rich

basal diet (ChB)

ChB+10% chitin

(deacetylated)

ChB+10% Shrimp

shell hydrolysate

(SSH)

Body mass index (BMI) 0.95 ± 0.04c 1.28 ± 0.05a 0.95 ± 0.05c 1.02 ± 0.04b

Average daily feed intake (g/day) 25.4 ± 1.4a 24.9 ± 1.5a 24.7 ± 1.7a 26.2 ± 1.6a

Average daily weight gain (g/day) 2.6 ± 0.09c 4.6 ± 0.21a 2.8 ± 0.08bc 3.1 ± 0.12b

Serum Triglycerides (mg/dL) 130.4 ± 5.6d 171.5 ± 8.2a 138.5 ± 5.7c 149.1 ± 5.3b

Serum Total cholesterol (TC) (mg/dL) 193.3 ± 9.1d 378.8 ± 15.5a 220.6 ± 9.1c 253.7 ± 10.3b

Serum HDL-cholesterol (HDL-C) (mg/dL) 50.7 ± 1.8a 50.1 ± 1.3a 53.7 ± 2.2a 52.4 ± 2.3a

Serum LDL-cholesterol (LDL-C) (mg/dL) 118.8 ± 2.5d 294.5 ± 4.5a 136.4 ± 3.2c 175.3 ± 3.1b

Atherogenic index (TC/HDL-C) 3.81 ± 0.13c 7.56 ± 0.21a 4.11 ± 0.16c 4.84 ± 0.10b

Total liver weight (LW) (g) 10.4 ± 0.5c 15.9 ± 0.6a 10.9 ± 0.5b 11.2 ± 0.5b

Relative LW (g/100 g body weight) 5.5 ± 0.3b 6.7 ± 0.3a 5.7 ± 0.3b 5.8 ± 0.3b

Liver Total lipids (mg/g of liver) 116.1 ± 3.3c 156.7 ± 4.7a 128.2 ± 3.2b 132.7 ± 3.6b

Liver Total cholesterol (mg/g of liver) 34.1 ± 1.5c 59.7 ± 2.3a 40.5 ± 2.3b 42.2 ± 3.3b

Liver Triglycerides (mg/g of liver) 57.1 ± 2.2c 75.2 ± 3.1a 63.2 ± 2.4b 65.1 ± 2.2b

Values within a row followed by different lower case letters are significantly different (P<0.05) according to ANOVA (Duncan’s

multiple range test)


929

arrangement (Fig. 4h). In contrast, supplementation

of cholesterol and cholesterol with chitin exhibited

negative impact on small intestinal structure

by decreasing the villus:crypt ratio with increasing

the crypt cells per villus as well as decreasing

the microbial colonization and atrophy in the

intestinal lining in comparison to the control group

(Fig. 4d and f).

Fig. 2—Effect of different diet schedule on the lipid peroxidation and antioxidant enzymes in male albino rats after 28 days of treatment;

A, B and C are the profile of MDA, SOD and CAT in serum, D, E and F are the profile of MDA, SOD and CAT in liver, G, H and I are

the profile of MDA, SOD and CAT in kidney respectively. ….. (dotted line) indicates the level of the respective parameters in control

group. *indicates the significant increase the respective parameter with respect to control group at P<0.05. **indicates the significant

decrease in the respective parameter with respect to control group at P<0.05


930

Fig. 3—Effect of different diet schedule on the growth of dominant intestinal microflora in male albino rats after 28 days of treatment. A

and B, C and D, E and F, G and H are the 4 dominant microbial profile of small and large intestine in control, cholesterol rich, cholesterol

rich+chitin, cholesterol rich+SSH diet group respectively. …..(dotted line) indicates the base GDI. *indicates the significant expansion of

the population (in GDI) at P<0.05. **indicates the significant contraction of the population (in GDI) at P<0.05

Table 2—Effect of different diet schedule on the fecal lipid profile in male albino rats after 28 days of treatment

[Values are mean ± SD from 20 animals in each group of triplicate determinations]

Parameters (mg/g of feces) Control ChB ChB+chitin ChB+SSH

Total lipids 68.5 ± 2.5d 78.3 ± 2.8c

93.8 ± 3.3a 86.9 ± 2.9b

Total cholesterol 15.4 ± 0.9c 18.7 ± 0.9b

31.5 ± 1.8a 27.7 ± 1.9a

Triglycerides 18.5 ± 1.1c 23.3 ± 1.1b

36.3 ± 1.7a 32.8 ± 2.1a

Diarrheal score (DS) 2c 13a

6b 3c

Values within a row followed by different lower case letters are significantly different (P<0.05) according to ANOVA (Duncan’s

multiple range test)


931

Discussion The degradation of crustacean biowaste by the

chitinolytic bacteria is the cost effective alternative

for the production of COS in large scale. A potent

chitinolytic bacteria Aeromonas hydrophila SBK1

was employed which utilized shrimp shell chitin as

sole carbon source and inducibly produced

chitinolytic enzymes during solid state fermentation

which subsequently catalyzes the degradation of

chitin into chitooligosaccharides (COS) having

differential degree of polymerization (DP) and free

radical scavenging activity. Many researchers have

focused on chitin and chitosan (deacetylated chitin) as

a potential bioactive material during past few decades.

However, they have several shortcomings to be

utilized in biological applications, including poor

solubility under physiological conditions. In this

study, we assessed the bioactivity efficiency of both

deacetylated chitin and chitooligosaccharides (COS).

Cholesterol rich feeding for 4 weeks increased

the BMI, serum and liver total cholesterol,

triglycerides, serum LDL-cholesterol, liver total lipid,

which collectively prompted the chance of

hypercholesterolemia associated diseases. Lu et al.27

have demonstrated that dietary cholesterol induced an

increase in the liver total cholesterol and liver free

Fig. 4—Effect of different diet schedule on the intestinal morphology in male albino rats after 28 days of treatment, A and B, C and D, E

and F, G and H are the histological and scanning electron microscopic (SEM) analysis of small intestine of rats of basal (control),

cholesterol rich, cholesterol rich+chitin, cholesterol rich+SSH diet group respectively. Arrows in SEM pictures indicating the position of

inflammatory sores and atrophy


932

cholesterol level. On the other hand, supplementation

of deacetylated chitin and SSH markedly lowerd the

said parameters still during cholesterol enriched

feeding along with the excretion of unabsorbed fat

through fecal matter. The cholesterol lowering

consequence may be due to the induced expression of

liver LDL-receptor which eliminates LDL-cholesterol

through endocytosis in liver by chitin and COS28

.

Scavengers of free radicals like ROS are preventive

antioxidants and presence of these compounds can

break the oxidative sequence at different levels.

Relationship between hypercholesterolemia and

oxidative stress has been extensively investigated;

direct evidence regarding the roles of cholesterol

accumulation in the generations of reactive oxygen

species was reported11

. Based on the results obtained

from the study, COS not only able to scavenge the

free radicals of the body but also trigger the enhanced

synthesis of CAT and SOD as well as minimize lipid

peroxidation. COS have shown potential as

scavenging agents, due to their ability to abstract

hydrogen atoms from free radicals29,30

. This ability

has been directly correlated with their structural

properties – namely that the amino and hydroxyl

groups can react with unstable free radicals to form

stable macromolecule radicals8,31

. Based on the results

obtained by Je et al.31

COS with low molecular

weight (1-3 kDa) have been identified to have a

higher potential to scavenge different free radicals.

Due to more available reactive groups and water

solubility, COS is superior over chitin. This is due to

the fact that unlike its high molecular weight

precursor, COS are easily absorbed through the

intestine, quickly get into the blood flow and have a

systemic biological effects in the organism.

Oligosaccharides are usually defined as prebiotics

which selectively stimulate the growth of health-

promoting bacteria, and as a consequence have a

beneficial impact on host health2. Thus, Lactobacillus

and Bifidobacterium are deemed target organisms

because of their potential to inhibit the growth of

putrefactive and pathogenic bacteria like Clostridium

perfringens and E. coli32

. In this study, dietary

supplementation of COS stimulates the growth of

Bifidobacter and Lactobacillus as well as decreased

the counts of Clostridium perfringens in both small

and large intestine. The exact mechanism through

which COS may modulate the growth of intestinal

bacteria remains uncertain. This is may be due to the

utilization of the COS as a growth promoter for

Bifidobacterium, Lactobacillus33

. In addition, COS

has been shown to be effective in inhibiting the

growth and activity of pathogenic bacteria, even

though those results were largely dependent on the

molecular weight of COS used in the several

studies34,35

. In the present study, COS having varying

DP (upto 30) with different molecular weight

(upto 5 kDa) was used, which has been shown to

modulate the immune response effectively and reduce

the onset of pathogenesis in the gut. It is also

well documented that prebiotics skip the

digestion/absorption in the small bowel and latter

fermented by probiotic bacteria in the colon which

ultimately helped in the production of short chain

fatty acids (SCFA) like acetate, propionate, butyrate

etc. which directly helped to reduced risk of some

deadly diseases36,37

. In agreement with present

findings, Li et al.38

reported that dietary COS

supplementation improved the gastrointestinal

Lactobacillus population. The present results were

also comparable with other studies3,39

. All these shifts

in the population of Bifidobacterium, Lactobacillus

and Clostridium indicated that COS may act as a

dietary prebiotic ingredient.

On the contrary, deacetylated chitin showed strong

antibacterial effect against colonic bacteria and

disrupts the gastrointestinal microbial ecology. Unlike

fully acetylated chitin, its partially or fully

deacetylated form possesses primary amino groups in

their structures in a row. The number of these amino

groups has proven to play a major role in antibacterial

activity40

. The mostly accepted mechanism explains

that deacetylated chitin can alter the permeability of

microbial cell membrane which subsequently causes

leakage which leads to death of bacteria41

.

Rats fed cholesterol rich diet had a dramatic

decline in Bifidobacter and Lactobacillus counts, an

intensive augmentation of the Clostridium perfringens

and E. coli population, and coincidently displayed a

greater incidence of diarrhea with greater diarrhea

scores as well as villus atrophy and formation of sore

on the intestinal wall. Conversely, COS efficiently

stimulate the probiotic bacterial growth, facilitate

elimination of unabsorbed lipid and cholesterol

through feces, decrease diarrheal score as well as

improve villus structure and villus:crypt ratio. Though

chitin have strong lipid and cholesterol binding ability

and eliminate them efficiently, its performance to

maintain the overall health status is impure than COS.

Therefore, it is theorized that the bioactivities of COS


933

and chitin differs in respect to their DD and DP.

From the present study it is also theorized that

chitooligosaccharides cocktails is better as a whole

than its precursor polymeric form (chitin) as it imparts

strong beneficial health impact.

Conclusion

The degree of polymerization, degree of

deacetylation and water solubility is the crucial

determinant of bioactivity of chitooligosaccharides

and chitin. Non-toxicity, biodegradability and

biocompatibility of COS promote their biological

applications compared to their precursor polymers.

The chitooligosaccharides cocktails of the fermented

end products of shrimp shells have strong cholesterol

lowering effect, antioxidant activity, can resist lipid

peroxidation and specifically influence the growth of

health beneficial microbes, hence can be implemented

as functional food for hypercholesterolemic patient

suffering in oxidative stress.

Acknowledgement Authors are grateful to Department of Science and

Technology (DST), Government of India for financial

assistance (INSPIRE Fellowship) and the authorities

of Vidyasagar University for infrastructural facilities.

References 1 Barreteau H, Delattre C & Michaud P, Production of

oligosaccharides as promising new food additive generation,

Food Technol Biotechnol, 44 (2006) 323.

2 Gibson G R & Roberfroid M B, Dietary modulation of the

human colonic microbiota: Introducing the concept of

prebiotics, J Nutr, 125 (1995) 1401.

3 Liu P, Piao X S, Kim S W, Wang L, Shen Y B, Lee H S & Li

S Y, Effects of chito-oligosaccharide supplementation on the

growth performance, nutrient digestibility, intestinal

morphology, and fecal shedding of Escherichia coli and

Lactobacillus in weaning pigs, J Anim Sci, 86 (2008) 2609.

4 Pluske J R, Thompson M J, Atwood C S, Bird P H, Williams

L H & Hartmenn P E, Maintenance of villus height and crypt

depth, and enhancement of disaccharide digestion and

monosaccharide absorption, in piglets fed on cows’ whole

milk after weaning, Braz J Nutr, 76 (1996) 409.

5 Boudry G, Péron V, Huërou-Luron I, Lallès J P & Sève B,

Weaning induces both transient and long-lasting

modifications of absorptive, secretory, and barrier properties

of piglet intestine, J Nutr, 134 (2004) 2256.

6 Durrington P, Dyslipidaemia, The Lancet, 362 (2003) 717.

7 Kontush A & Chapman M J, Antiatherogenic small, dense

HDL--guardian angel of the arterial wall?, Nat Clin Pract

Cardiovasc Med, 3 (2006) 144.

8 Kim S K & Rajapakse N, Enzymatic production and

biological activities of chitosan oligosaccharides (COS):

A review, Carbohydr Polym, 62 (2005) 357.

9 Uttara B, Singh A V, Zamboni P & Mahajan R T, Oxidative

stress and neurodegenerative diseases: a review of upstream

and downstream antioxidant therapeutic options,

Curr Neuropharmacol, 7 (2009) 65.

10 Sharma N & Garg V, Antihyperglycemic and antioxidant

potential of hydroalcoholic extract of Butea monosperma

Lam flower in alloxan-induced diabetic mice, Indian J Exp

Biol, 47 (2009) 571.

11 Lee W, Xu M, Li Y, Gu Y, Chen J, Wong D, Fung P C &

Shen J, Free cholesterol accumulation impairs antioxidant

activities and aggravates apoptotic cell death in menadione-

induced oxidative injury, Arch Biochem Biophys,

514 (2011) 57.

12 Kandra P, Challa M M, Padma Jyothi H K, Efficient use of

shrimp waste: present and future trends, Appl Microbiol

Biotechnol, 93 (2012) 17.

13 Okamoto Y, Inoue A, Miyatake K, Ogihara K, Shigemasa Y

& Minami S, Effects of chitin/chitosan and their oligomers/

monomers on migrations of macrophages, Macromol Biosci,

3 (2003) 587.

14 Rhoades J, Gibson G, Formentin K, Beer M & Rastall R,

Inhibition of the adhesion of enteropathogenic Escherichia

coli strains to HT-29 cells in culture by chito-

oligosaccharides, Carbohydr Polym, 64 (2006) 57.

15 Halder S K, Maity C, Jana A, Pati B R & Mondal KC,

Chitinolytic enzymes from the newly isolated Aeromonas

hydrophila SBK1: study of the mosquitocidal activity, Bio

Control, 57 (2012) 441.

16 Wang S L, Lin C L, Liang T W, Liu K C & Kuo Y H,

Conversion of squid pen by Serratia ureilytica for the

production of enzymes and antioxidants, Bioresour Technol,

100 (2009) 316.

17 Carlson S E & Golgfard S, A sensitive enzyme method for

determination of free and esterified tissue cholesterol,

Clin Chem Acta, 79 (1979) 575.

18 Folch J, Lees M & Stoane-Stanley G H, A simple method for

the isolation and purification of total lipid from animal tissue,

J Biol Chem, 223 (1957) 497.

19 Aebi H E, Catalase in Methods in enzymatic analysis edited

by HU Bengmeya, (Verlag Chemie Weinhien, Germany)

1983, 278.

20 Marklund S & Marklund G, Involvement of the superoxide anion

radical in autoxidation of pyrogallol as a convenient assay for

superoxide dismutase, Euro J Biochem, 47 (1974) 469.

21 Lowry O H, Rosebrough N J, Farr A L & Randall R J,

Protein measurement with the folin phenol reagent,

J Biol Chem, 193, (1951) 265.

22 Buege J A & Aust S D, Microsomal lipid peroxidation,

Methods Enzmol, 52 (1978) 302.

23 Maity C, Pathak T K, Pati B R, Adak A & Mondal K C,

Study of the cultivable microflora of the large intestine of the

rat under varied environmental hyperbaric pressures,

J Microbiol Immunol Infect, 45 (2012) 281.

24 Hart G K & Dobb G J, Effect of a fecal bulking agent on

diarrhea during enteral feeding in the critically ill, J Parenter

Enteral Nutr, 12 (1988) 465.

25 Montagne L, Cavaney F S, Hampson D J, Lalle J P & Pluske

J R, Effect of diet composition on postweaning colibacillosis

in piglets, J Anim Sci, 82 (2004) 2364.

26 Li D F, Thaler R C, Nelssen J L, Harmon D L, Allee G L &

Weeden T L. Effects of fat sources and combinations on

starter pig performance, nutrient digestibility and intestinal

morphology, J Anim Sci, 68 (1990) 3694.


934

27 Lu Y F & Wu H L, Effect of monounsaturated fatty acids

under fixed P/S and n-6/n-3 ratios on lipid metabolism in

rats, J Nutr Sci Vitaminol, 40 (1994) 189.

28 Chiang M T, Yao H T & Chen H C, Effect of

dietary chitosans with different viscosity on plasma

lipids and lipid peroxidation in rats fed on a diet enriched

with cholesterol, Biosci Biotechnol Biochem, 64 (2000)

965.

29 Huang R, Mendis E & Kim S K, Factors affecting the free

radical scavenging behavior of chitosan sulfate, Int J Biol

Macromol, 36 (2005) 120.

30 Varum K M, Ottøy M H & Smidsrød O, Water-solubility of

partially N-acetylated chitosans as a function of pH, effect of

chemical composition and depolymerisation, Carbohydrate

Polym, 25 (1994) 65.

31 Je J Y, Park P J & Kim S K, Free radical scavenging

properties of heterochitooligosaccharides using an ESR

spectroscopy, Food Chemical Toxicol, 42, (2004) 381.

32 Paton A W, Morona R & Paton J C, Designer probiotics for

prevention of enteric infections, Nat Rev Microbiol, 4 (2006)

193.

33 Lee H W, Park Y S, Jung J S & Shin W S, Chitosan

oligosaccharides, dp 2-8, have prebiotic effect on the

Bifidobacterium bifidum and Lactobacillus sp, Anaerobe, 8

(2002) 319.

34 Matsuhashi S & Kume T. Enhancement of antimicrobial

activity of chitosan by irradiation, J Sci Food Agric, 73

(1997) 237.

35 Tsai G J, Wu Z Y & Su W H, Antibacterial activity of a

chitooligosaccharide mixture prepared by cellulose digestion

of shrimp chitosan and its application to milk preservation, J

Food Prot, 63 (2000) 747.

36 Roediger W E, Role of anaerobic bacteria in the metabolic

welfare of the colonic mucosa in man, Gut, (1980) 793.

37 Floch M H & Rowland I R. Probiotics and functional foods

in gastrointestinal disorders, Current Treat Options

Gastroenterol, (2002) 311.

38 Li X J, Piao X S, Kim S W, Liu P, Wang L, Shen Y B, Jung

S C & Lee H S, Effects of chito-oligosaccharide

supplementation on performance, nutrient digestibility, and

serum composition in broiler chickens, Poult Sci, 86 (2007)

1107.

39 Pluske J R, Hampson D J & Williams I H, Factors

influencing the structure and function of the small intestine

in the weaned pig: A review, Livest Prod Sci, 51 (1997) 215.

40 Chen Y M, Chung Y C, Wang L W, Chen K T & Li S Y,

Antibacterial properties of chitosan in waterborne pathogen,

J Environ Sci Health, 37 (2002) 1379.

41 Sudharshan N R, Hoover D G & Knorr D, Antibacterial

action of chitosan, Food Biotechnol, 6 (1992) 257.

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