CHITIN AND CHITOSAN FROM SHRIMP WASTE BY

CHEMICAL AND MICROBIAL METHOD

Chitin

Chitin is a versatile environmentally friendly modern material (Mahmoud, 2007). It is a naturally

occurring high molecular weight linear homopolysaccharide composed of N-acetyl-D
glucosamine residues in (1-4) linkage. Chitin and chitin derivatives are biodegradable and
biocompatible natural polymers that have been used in virtually every significant segment of the
economy (e.g. water treatment, pulp and paper industry, biomedical devices and therapies,
cosmetics, biotechnology, agriculture, food science and membrane technology) (Li et al., 1997).
Chitin can be found in a variety of species in both the animal and plant kingdoms. The traditional
source of chitin is shellfish waste from shrimp, Antarctic Krill, crab and lobster processing
(Muzzarelli, 1977; Shahidi, 1991). Chitosan is a natural, non-toxic, co-polymer of glucosamine
and N-acetylglucosamine obtained after partial de-N-acetylating of chitin, which, in turn, is a
major component of the shells of crustaceans and found commercially in the offal of marine food
processing industry (Tharanathan and Kitture, 2003). In spite of its abundance and various
biofunctionalities, utilization of chitosan is restricted, owing to its high molecular mass, high
viscosity and, thus, low absorption for in vivo applications (Shon, 2001). Recent studies on
chitosan depolymerization have drawn considerable attention, since the products obtained are
easily water soluble and also possess versatile biofunctional properties such as antitumour
(Suzuki, 1986; Ikeda, 1995), immuno-stimulating (Shon, 2001) and antimicrobial activities
(Sekiguchi, 1994; Jeon, 2001) and are being used for alleviating problems due to
osteoarthritisgastritis (Sukwattanasinitt, 2002).
In traditional chemical methods for isolating chitin from shrimp waste, 4% NaOH is used for
deproteination and 4% HCl for demineralization. This process may not be considered as a good
recovery option, because it is expensive and non-environmentally friendly (Rao, 2002). Partial
fermentation of this biowaste using lactic acid bacteria for the production of chitin has been
studied and reported (Rao, 2000).

The objective of this research was to study the effect of three strains of lactic acid bacteria on the
fermentation efficiency of shrimp biowaste to recovery chitin and chitosan, its comparison with
chemical extraction method and also studying the effects of intervening factors on microbial
extraction.

MATERIALS AND METHODS

Shrimp waste
Shrimp waste from processing of Penaeus semisulcatus, comprising of head and carapace, was
collected from a shrimp processing landing center situated at Persian Gulf in south of Iran. These
shrimp were caught in the fall of 2007. Following cooking in boiling salt water for 10 minutes,
the shrimp were sent to automated peeling machines, where the shell and meat portions were
separated. The shell material was collected and dried at 50C in oven for 24h and homogenized
in a laboratory mixer before shipping for further processing. The yield of dried shell was
determined by weighting after being dried. The obtained shrimp shells were stored at about-25C
in the storage facility till needed.
Chemical extraction of chitin and chitosan
The process of extraction involved deproteinization with 2% w/v sodium hydroxide solution
(30:1 v/w, 90C, 2h), separation of alkali-insoluble fraction (AIF) by centrifugation (4000rpm,
15min.), extraction of chitosan from AIF under reflux (10% v/v acetic acid 40:1 v/w, 60C, 6h),
separation of crude chitin by centrifugation (4000rpm, 15min) and precipitation of chitosan from
the extract at pH=9, adjusted with a 4M NaOH solution. Crude chitin and chitosan were washed
on a coarse sintered-glass funnel with distilled water, ethanol and acetone and air-dried at 20C
(Synowiecki, 1997).

Moisture content
Crude chitin sample was placed in a pre-weighted aluminum dish. The dish and contents were
then placed in an oven at 105C for 24h. The aluminum dish along with the dried sample was
first placed in a desicator to cool down and then weighted. The moisture content was determined
as follows (Mahmoud, 2007):
Chitin minerals
The demineralized shell material (purified chitin) was filtered under suction condition. The
recovered solids were thoroughly washed using deionized-distilled water and dried in an oven at
60C for 24h. The dried purified chitin samples were analyzed for their mineral content.
Quantitative trace element analyses were done using an atomic absorption spectrophotometer.
For Calcium, manganese, iron and copper analyses, the samples were first digested with
hydrochloric and nitric acids (30, 10mL/g sample, respectively) in a closed vessel at a
temperature of 100C and then the elements were determined by flame atomic absorption with
detection limit of 1 ppm (Mahmoud, 2007).
Microorganism and culture media
Three species of Lactobacillus named Lactobacillus plantarum (PTTC 1058), Lactobacillus
acidophilus (PTTC 1643) and Lactobacillus rhamnosus (PTCC 1637) were provided from
Persian Type Culture Collection (PTCC) of Iranian Research Organization for Science and
Technology in Tehran, Iran. They were sub cultured on MRS broth and agar media (10g peptone
from casein, 4g yeast extract, 8g meat extract, 20g D(+) glucose, 1g tween 80, 2g di-ammonium
hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate
MERCK). MRS Broth was mixed with 15g/L agar to solidify the medium and incubated at 3537C in the presence of 5% CO2 for 48-72h.
Microbial extraction of chitin and chitosan
After adding 5 mL of MRS broth containing each of the three Lactobacillus spp. (OD600=0.8-1)
to the fermentative medium culture (50mL distilled water +5g of shrimp waste powder) samples

were incubated for 7 days at 30C, in the presence of 5% CO2. The fermentative culture medium
was centrifuged at 3000rpm for 5min. The recovered solids were washed thoroughly several
times using deionized-distilled water. Chitin and chitosan were extracted with the same method
described previously.
Optimization of conditions for microbial extraction of chitin and chitosan
The conditions for extraction were optimized with the effect of a combination of process
variables (factors) such as lactose sugar, yeast extract and Fe (NO 3)3 1% was added to
fermentation medium and their interactions on the response variable were determined. The
extraction of chitin and chitosan and the determination of their concentration were carried out as
explained earlier.
RESULTS
Result of chitin and chitosan yields in chemical extraction are present in Table 1. No significant
difference

was observed in chitin yields

plantarum (PTTC

1058), Lactobacillus

in microbial

acidophilus (PTTC

extraction

by Lactobacillus

1643)

and Lactobacillus

rhamnosus (PTCC 1637) (P<0.05). But Lactobacillus plantarum (PTTC 1058) ability to extract
chitosan was more than two other bacteria (Table 2). This was the same as chitosan yield
extracted by chemical method. The microbial method could extract chitin better than chemical
method (Table 1, 2).
Chitin and chitosan extracted from shrimp waste by chemical and microbial methods was
crystalline powder, non-harmful and odorless and were white and off-white, respectively.
The moisture content was calculated as 63.8%. The amount of Ca, Fe, Cu and Mn present in the
shrimp waste was 168, 35.58, 38.28 and 6.72mg/L, respectively. The amount of calcium present
in the shrimp waste was 25 times higher than the amount of manganese.
Optimization of conditions for chitin and chitosan extraction showed that, when Fe (NO3)3, as a
mineral nitrogen source, was added to fermentation medium, it gave a higher chitin yield

comparing with adding yeast extract (as organic nitrogen source) as at least 810 and 1750mg/g,
respectively. But it did not have a significant increasing effect on the chitosan yield (Fig.1).
Results obtained from this study showed that the increasing lactose to fermentation broth, had an
amplifying effect on the extraction of chitin and chitosan yield, the same as the case of yeast
extract was added (Fig.1).
DISCUSSION
Extraction of chitin and chitosan from a certain Persian Gulf shrimp species waste (Penaeus
Semisulcatus), by chemical and microbial methods was investigated. In this study, the results
showed that, microbial method is more effective especially for the recovery of chitin comparing
with chemical method. Overally, extraction of chitin from biowaste by three strains was equal.
The growth and lactic acid production by these bacteria increased the products. The strain 1058
showed higher fermentation efficiency, especially in chitosan yield extraction (32.25%).
According to Allan, et al., 1978, the traditional method involving the use of strong acids makes
this process ecologically aggressive and a source of pollution. It also reduces a certain degree of
depolymerization and thus chitin quality (Hansen, 1994; Simpson, 1994). The chemical
treatment renders the protein component useless, which otherwise can be used as animal feed.
Percot et al., (2003) reported that using inorganic acid such as HCl for the demineralization of
chitin, results in detrimental effects on the molecular weight and the degree of acetylating that
negatively affects the intrinsic properties of the purified chitin.
For microbial extraction, authors used three strains of lactic acid bacteria. Using organic acids
such as lactic acid for the demineralization process is a promising idea since organic acids can be
produced with low cost by bacteria, are less harmful to the environment, can preserve the
characteristics of the purified chitin and the resulting organic salts from the demineralization
process can be used as an environmentally friendly deicing/anti-icing agents and/or as
preservatives. Fagbenro, 1996, used raw heads of Africa river prawn (Macrobrachium
vollenhovenii) fermented with Lactobacillus plantarum using cane molasses. Lactic acid
bacterial fermentation for demineralization has been occasionally reported for shrimp waste

(Shirai, 2001), crayfish exoskeleton (Bautista, 2001), scampi waste (Zakaria, 1998), and prawn
waste (Shirai, 1998).
Fe(NO3)3 as a mineral nitrogen source, gave a higher chitin yield than added yeast extract (as
organic nitrogen source) or lactose (as a carbon source). The most significant effect was due to
Fe(NO3)3 (1750mg/g), after and then those of lactose (820mg/g), and yeast extract (810mg/g).
Hall and Silva (1992) studied lactic acid bacterial fermentation of shrimp waste (Penaeus
monodon) for chitin recovery with added carbohydrate such as lactose or cassava extract as a
natural energy source. Treatment of minced scampi (Nephrops norvegicus) waste (supplemented
with glucose) by a culture of Lactobacillus paracasei strain A3 was investigated by Zakaria et
al.,(1998). The efficiency of demineralization was affected by the various inoculum amounts
supplied. Also, the proportions of the additional starter and glucose were important for the lactic
acid bacterial fermentation to demineralize the raw shell wastes (Meraz, 1992; Shirai, 2001; Rao,
2002).
Jung et al., showed that lactic acid fermentation resulted in a partial deproteination from
the red crab shell waste, since the bacterium produced proteases. About 40% and 30% of proteins
were removed in 5% and 10% inoculums, respectively, with 10% glucose after 5 days of
fermentation. The lactic acid reacts with the calcium carbonate component in the chitin fraction,
leading to the formation of calcium lactate, which precipitates and can be removed by washing.
Woods, 1998 demonstrated that, deproteination of the biowaste and simultaneous liquefaction of
the shrimp proteins occurs mainly by proteolytic enzymes produced by the added lactobacillus,
by gut bacteria present in the intestinal system of the shrimp, or by proteases present in the
biowaste.
Morita et al. 1997, showed that, Lactobacillus fermentum IFO 3956 that differed in origin
converted metmyoglobin to nitrosylmyoglobin (a pentacoordinate nitric oxide (NO) complex of
Fe (II) myoglobin) in MRS broth at pH=4.3. Xu and Verstraete, 2001, investigated on six strains
of Lactobacillus fermentum and Lactobacillus plantarum for nitric oxide (NO) production.
All Lb. fermentum strains produced NO in MRS broth, but the NO was found to be chemically
derived from nitrite, which was produced by Lb. fermentum from nitrate present in the medium.
Indeed all Lb. fermentum strains express nitrate reductase under anaerobic conditions. NO and

nitrite produced from nitrate by lactobacilli may constitute a potential antimicrobial mechanism.
Thereby, NO3 may play an acceleration role for Lactobacillus growth.
Results of this study showed that, increasing lactose to fermentation broth, had an amplifying
effect on the extraction of chitin (820mg/g) and chitosan (500mg/g) yield.
The influence of carbon and nitrogen sources on chitin production by microorganisms
was evaluated with a factorial design analysis (Andrade, 2000). It was found that chitin
production was affected mostly by L-asparagine, followed by D-glucose and thiamine. The value
of mineral content of the shrimp shells used in this study was within the range of 6.72-168mg/L.
The most abundant minerals in the shrimp waste were calcium. Hansen and Illanes (1994) stated
that the major mineral component of shellfish waste is calcium. Beaney et al., (2005), reported
that the most abundant minerals in prawn shell were Ca, Mg, Na, Sr, K and Fe in that order and
that calcium was by far the most abundant (about 17 times more than magnesium). Synowiecki
and Al-Kateeb (2003) stated that the minerals fraction of shrimp shells composed mostly of
phosphates and carbonates of calcium and magnesium. Mahmoud et al. (2007), reported, the
amount of calcium present in the shells was 6 and 23 times higher than the amounts of
phosphorus and magnesium, respectively. Manganese yield in shrimp waste was not determined
by these scientists. Results of the manganese yield in Penaeus semisulcatus waste in our study
was similar the magnesium yield determined by Mahmoud et al., (2007).
The effectiveness of lactic acid (produced by lactic acid bacteria) in removing the
minerals from the shells and extracting chitin was higher than sulfuric acid in chemical method,
so lactic acid fermentation could provide an alternative to chemical treatment, to extraction and
recovery of chitin and its derivates.

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