Chitin extraction from shrimp (Litopenaeus vannamei) shells by successive two-step fermentation with Lactobacillus rhamnoides and Bacillus amyloliquefaciens

Journal Pre-proof Chitin extraction from shrimp (Litopenaeus vannamei) shells by successive two-step fermentation with Lactobacillus rhamnoides and Bacillus amyloliquefaciens

Yongliang Liu, Ronge Xing, Haoyue Yang, Song Liu, Yukun Qin, Kecheng Li, Huahua Yu, Pengcheng Li PII:

S0141-8130(19)39715-6

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.124

Reference:

BIOMAC 14428

To appear in:

International Journal of Biological Macromolecules

Received date:

27 November 2019

Revised date:

13 January 2020

Accepted date:

13 January 2020

Please cite this article as: Y. Liu, R. Xing, H. Yang, et al., Chitin extraction from shrimp (Litopenaeus vannamei) shells by successive two-step fermentation with Lactobacillus rhamnoides and Bacillus amyloliquefaciens, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.01.124

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© 2018 Published by Elsevier.

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Chitin extraction from shrimp (Litopenaeus vannamei) shells by successive two-step

fermentation

with

Lactobacillus

rhamnoides

and

Bacillus amyloliquefaciens Yongliang Liu

a,b,c

, Ronge Xinga,b*, Haoyue Yanga,b, Song Liua,b, Yukun Qina,b, Kecheng

Lia,b, Huahua Yu a,b, Pengcheng Lia,b* a

Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science,

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine

of

b

University of Chinese Academy of Sciences, Beijing 100049, China.

-p

c

ro

Science and Technology(Qingdao), No. 1 Wenhai Road, Qingdao 266237, China;

re

Abstract

fermentation.

The

best

microorganisms

Lactobacillus

na

two-step

lP

Chitin was extracted from shrimp shells powders (SSP) by successive

rhamnoides and Bacillus amyloliquefaciens (BA01) for demineralization

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(DM) and deproteinization (DP) were obtained and the optimal fermentation conditions for two-step fermentation were established. Firstly, we determined the cultured conditions (inoculum level 4%, initial pH 6.5, cultured temperature 37 ℃, glucose concentration 5%, cultured time 48 h) of Lactobacillus rhamnoides and the organic acid quantities and types of fermentation broth of Lactobacillus rhamnoides. Under the conditions, the pH of fermentation broth was 3.4，the DM efficiency was 97.5% and the ash in the final residue was 1.2%, and the main organic acid was lactic acid. Secondly, the optimal cultured conditions of BA01 1

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were inoculum level 6%, initial pH 6.5, cultured temperature 37 ℃, glucose concentration 4%, and cultured time 84 h. Under the conditions, the protease activity of fermentation broth was 701.3 U/mL, the DP efficiency was 96.8%, the protein in the final residue was 1.5%, and the chitin yield was 19.6%. In addition, the chitin obtained by fermentation

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was compared with the commercial chitin using scanning Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD),

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Thermogravimetric analysis (TGA), Solid-state

13

C CP/MAS-NMR

chitin

obtained

by

fermentation

re

the

-p

spectra, and Scanning electron microscope (SEM). The results showed maintains

the

excellent

lP

physicochemical and structural properties of commercial chitin.

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Moreover, in order to make full use of shrimp and crab shells resources, the amino acid composition of fermentation broth was detected. The

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results showed that the fermentation broth had high nutritional value and could be used as a health nutrient in animal feed, even food. Keywords:

Shrimp

shells,

two-step

fermentation,

Lactobacillus

rhamnoides, Bacillus amyloliquefaciens, chitin 1. Introduction Processing of large bulk of crustaceans such as shrimp and crab will

produce a large number of by-products and wastes (heads, shells, etc.). Crustaceans wastes management is a huge problem, especially for crustacean shells, which lack cost-effective waste exports, a considerable amount of waste was discharged as processing wastewater [1]. Because 2

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of their high availability and their chemical composition (15–25% chitin, 35–50% protein, and 25–35% minerals), the shells of crustaceans are the most important source of commercial chitin. The potential use of chitin and chitosan is widely recognized, e.g., in agriculture, food technology, cosmetics industry, chemical industry, medical industry, pharmaceutical

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industry, biomedical industry, and other industries [2, 3]. At present, methods for extracting chitin from shrimp shells waste mainly include

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chemical methods and biological methods, moreover, chemical methods

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have been used for large-scale preparation of chitin. In chemical methods,

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chitin was extracted from crustacean shells using a large number of

lP

strong acid (HCl) and bases (NaOH) to remove minerals, protein, and

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lipids, the application of strong acids and bases may lead to hydrolysis of the polymer, and inconsistencies in the physical properties of chitin.

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Moreover, the use of chemical agents will make an aggressive source and pollute the ecological environment, and harm human and animal health [4]. Today, with the development of environmental technology concepts, an environmentally friendly method for large scale extraction of chitin needs to be developed. A newer method for demineralization and deproteinization of crustacean shells using organic acids and protease producing bacteria was reported. The deproteinization process for the preparation of chitin from crustacean shells by enzymatic method has been reported [5, 6], and microbes such as Aquatic Bacillus sp, Serratia 3

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marcescens FS-3, the efficiency of DP was 80.17% and 68.9% [7, 8]. Also, researches on demineralization processes have also been reported for crustacean shells using lactic acid bacteria, and Gluconobacter oxydans, the efficiency of DM was 85.0%, 61.0% and 87.0%, respectively [9-11]. Since the efficiency of DP and DM was not high enough to obtain pure

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chitin, the organic reagents were subsequently used. Furthermore, chitin obtained from crustacean shells by fermentation has demonstrated to have

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better quality than chitin obtained by chemical methods [12]. The present

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study aimed to ferment and extract chitin from shrimp shells by two-step

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fermentation with Lactobacillus rhamnoides and BA01 strain. We try to

lP

completely replace the chemical method by fermentation to obtain pure

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chitin from shrimp shells, and conduct structural characterization of the products to ensure that the products have the same structure as

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commercial chitin. For efficient recovery of pure chitin, the cultured key conditions, including inoculum level, initial pH, cultured temperature, glucose concentration, and cultured time were optimized. The best fermentation conditions for two-step fermentation have been investigated as a promising method for processing. Lactobacillus rhamnoides strain was used for demineralization processes and BA01 strain which was isolated from shrimp intestine was used for deproteinization processes. 2. Materials and methods 2.1 Shrimp shells powders 4

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Shrimp (Litopenaeus vannamei) shells were obtained from Qingdao taidong aquatic products market (Shandong, China). The shells were washed and dried in an oven at 60 ℃ for 2 days, and then pulverized into a powder of 0.5-1.0 mm particle size and stored in the refrigerator at -20 ℃.

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2.2 Preparation of bacterial strains The lactic acid bacterium Lactobacillus rhamnoides was obtained from

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the China General Microbiological Culture Collection Center (CGMCC).

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The protease producing bacterium BA01 was isolated and identified from

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shrimp intestine. Briefly, approximately 0.1 g shrimp intestinal contents

lP

were prepared in solutions with different dilutions (10-1 - 10-9) using

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sterilized saline and ten-fold dilution method. Diluted solution (50 L) was applied onto a plate of LB medium. After incubation at 30 ℃ for 24

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h, plates with about 10 bacterial colonies were selected. The bacterial strains with high proteinase were screened according to the method in the literature [13]. Finally, the selected strain was identified by 16SrDNA. In order to prepare a starter culture, the Lactobacillus rhamnoides and BA01 were transferred into 100 mL of sterile Man Rogosa Sharpe (MRS) broth and LB broth, respectively, and incubated with shaking (150 rpm) at 30 ℃ for 2 days. For the separate fermentation, shrimp shells powders (5 g) were added to 100 mL of water supplemented with 3% glucose and inoculated with 2% Lactobacillus rhamnoides or BA01 at the beginning 5

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of fermentation in a shaking incubator (150 rpm) at 30 ℃ for 48 h. 2.3 Chemical composition analysis Ash content was determined by the weighing method. The sample was heated in a muffle furnace at 600°C for 300 min, and then cooling down in a desiccator for 10 min to ambient temperature, and balanced to

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calculate the percentage of residual weight. The pH of the supernatant during Lactobacillus rhamnoides and BA01

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fermentation was measured using a potentiometer (PHS-3C, Shanghai,

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China).

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Total nitrogen contents were measured by Kjeldahl’s method in an

lP

automated equipment (KDN-CZ, China). DM and DP percentages were

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expressed and computed by equation [14].

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DP (%) =

(PO × O) − (PR × R) × 100 PO × O

Where PO and PR were the protein contents (%) of original and residue samples respectively, and O, as well as R, represented the masses (g) before and after fermentation respectively. DM (%) was calculated using a similar equation, while PO and PR were replaced with AO and AR, which represented the ash content in the original and the residue samples, respectively.

DM (%) = 6

(𝐴𝑂 × 𝑂) − (𝐴𝑅 × 𝑅) × 100 𝑃𝑂 × 𝑂

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Protease activity was detected by the method described by Anraku et al. [15] using casein as a substrate. Briefly, 0.5 mL aliquot of the cultured supernatant was diluted and mixed suitably with 0.5 mL of 100 mM potassium phosphate buffer (pH 7.2) containing 1.0% (w/v) casein. The reaction mixture was then incubated for 15min at 50 ℃, completed by

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the addition of 0.5 mL of 1.0 mL 0.4 M trichloroacetic acid. The reaction mixture was allowed to sit at room temperature for 20 min and then

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centrifuged at 11,000 rpm for 15 min to remove the precipitate. The

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standard curve was generated using solutions of 0 mg/L, 10 mg/L 20

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mg/L, 30 mg/L, 40 mg/L, and 50 mg/L of tyrosine. The absorbance of the

lP

mixture was detected at 660 nm. One unit of protease activity was

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defined as the amount of enzyme required to release 1 gram of tyrosine per minute under the experimental conditions. The activity of protease

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represents the means of at least three determinations carried out in triplicate and the differences between values did not exceed 5%. 2.4 Quantification of organic acids The organic acid quantities and types of organic acid of fermentation broth of Lactobacillus rhamnoides (FBLA) were determined by RP-HPLC (LC-20 AD, Japan), using the method of Barbano et al. [16]. Citric acid, lactic acid, acetic acid, succinic acid, malic acid, and formic acid was used as standards. 7

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2.5 Quantification of total amino acids The

method

used

for

hydrolysis

was

mainly

according

to

Sánchez-Machado et al. [17]. Samples (50 mg) were transferred to in tubes and hydrochloric acid (6 M, 10 mL) was added, the nitrogen-filled tubes were placed in an oven 110 ℃ drying for 24 hours. The

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hydrolysate samples were then diluted to a concentration of 0.3mg/mL in

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50 mL volumetric flasks. The hydrolyzed samples (1 mL) were dried in a

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vacuum oven for 24 h. Next, the residues were dissolved in the sample

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buffer (1 mL, pH 2.2, citrate solution) and filtered through a 0.22μm

lP

membrane. Then the amino acids were analyzed using an automatic amino acid analyzer (Sykam, Germany).

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2.6 Characterization

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Fourier transform infrared (FT-IR) spectra were investigated by a FT-IR spectrometer (Thermo Scientific NicoletiS10, USA), And the operating conditions were 32-time scans, 4 cm-1 resolution with the spectral range from 4000 to 400 cm-1. X-ray diffraction (XRD) patterns were detected by an X-ray Diffractometer (BRUCKER D8, Germany). The XRD spectra was obtained with Cu Kα radiation at 50 kV and 40 mA. A continuous scan was performed using a step size of 0.02◦ and a step time of 0.2 s. Thermogravimetric analysis (TGA) was carried out using a METTLER 8

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TGA/DSC instrument (SF/1382, Germany) a heating rate of 10 ℃ / min under a nitrogen atmosphere. Solid-state

13

C CP/MAS-NMR spectra were performed on a Solid-State

NMR Spectrometer (Bruker AVANCE III 600 M, Germany) spectrometer with a time-domain size of 2048 and 1000 scans.

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Scanning electron microscope (SEM) was performed using a SU8020 (HITACHI, Japan). The samples were placed on a sample holder and a

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thin layer of gold was plated by ion sputtering to obtain conductivity.

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2.7 Statistical analysis

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Experimental data were statistically analyzed by one-way analysis of

lP

variance (ANOVA) using SPSS version 17.0 software (SPSS Inc.,

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Chicago, IL, USA). Significant differences between groups were tested by One-way ANOVA. Duncan's multiple range test was used for

0.05.

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individual comparisons. Statistical significance was determined at P <

3. Results and discussion 3.1 Strain identification

NCBI data showed that the homology between this screened strain (BA01) with high proteinase and Bacillus amyloliquefaciens was 99%. According to this result, the strain was identified as Bacillus amyloliquefaciens (data was not shown). 9

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3.2 Cultivation strategies by Lactobacillus rhamnoides and BA01 Three different cultivation strategies, namely B + L (simultaneous inoculation), B → L: first BA01 then Lactobacillus rhamnoides; L → B: first Lactobacillus rhamnoides then BA01, were employed as described above, and the changes of protein contents, ash contents, DP, and DM

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during fermentation were detected. As shown in Table 1, during the

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treatment of B + L, the efficiency of DM and DP was significantly lower

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(P < 0.05) than that of single cultivation. For DP, the two-step

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fermentation with B → L achieved a slightly lower (P > 0.05) removal efficiency (83.33%) than L → B (84.77%), while the DM efficiency of B

lP

→ L (80.92%) was significantly lower (P < 0.05) than L → B (84.85%).

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Jung et al. [18] extracted chitin by using the method of co-fermentation

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from red crab shells. They obtained DP and DM efficiency of 52.6% and 94.3%. Due to the mutual inhibition of the two strains, we have to take a two-step fermentation strategy of L → B. In this process, the strain that produced organic acid precedes the proteolytic strain because the DM process broke up the calcium-protein-chitin complex in the skeletal tissue, removed some minerals during the fermentation, which caused the microfibers in the shrimp shells to swell, thus facilitating the DP process. Although this may take more time, the DM efficiency was higher than the value cited in the literature above. In addition, both Lactobacillus rhamnoides and BA01 are safe organisms. According to the studies of 10

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Jung et al. [18], the strains they used were conditional pathogens, which do not meet the requirements of the food industry. Table 1 Changes of protein contents, ash contents, DP, and DM during

DP (%)

DM (%)

29.64 ± 1.34 22.08 ± 0.69a 3.35 ± 0.27b 15.31 ± 0.99c 3.45 ± 0.21b 3.04 ± 0.06b

32.36 ± 1.75 3.57 ± 0.36a 27.08 ± 1.66b 7.09 ± 0.66c 5.29 ± 0.23d 3.25 ± 0.17a

-30.50 ± 1.56a 83.28 ± 0.64c 48.28 ± 2.99b 83.33 ± 0.39c 84.77 ± 0.51c

-83.83 ± 0.72a 9.30 ± 1.73b 78.00 ± 1.51c 80.92 ± 1.12c 84.85 ± 0.55a

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Ash (%)

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Before L B B+L B→L L→ B

Protein (%)

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fermentation

The results are presented as the mean ± SD; a, b, c Mean values bearing

lP

different superscripts in a row differ significantly (P < 0.05). B + L:

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simultaneous inoculation; B → L: first BA01 then Lactobacillus

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rhamnoides; L → B: first Lactobacillus rhamnoides then BA01; DP (%) represented the efficiency of deproteinization; DM (%) represented the efficiency of demineralization. 3.3 Effect of carbon sources on DM and DP efficiency The present study showed that Lactobacillus rhamnoides could produce a large amount of organic acid and BA01 could produce high levels of protease activity when were cultured in media containing shrimp shells. During the fermentation of shrimp shells, the minerals were mainly removed by organic acids produced by Lactobacillus rhamnoides while 11

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the protein was mainly removed by proteolytic enzymes produced by BA01. The amount of organic acid and activity of proteases produced by microbial fermentation was affected by many factors, such as carbon source, inoculation concentration, pH, temperature, culture time, etc. We first studied the effects of several carbon sources such as sucrose, maltose,

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starch, glucose, and lactose on the DM and DP of two strains. Chien Thang et al. [19] demonstrated that external carbon sources added to

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shrimp wastes medium could promote microbial growth and acid

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production. Figure 1 showed that the addition of carbon sources to the

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medium increased the DM and DP efficiency, respectively. Significantly

lP

higher effect on DM and DP efficiency was both exhibited in treatments

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added with glucose as compared with other carbon sources (P < 0.05). This was probably due to both two strains had a higher ability to

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hydrolyze and metabolize glucose monomer than other carbon sources. Hence, further studies were carried out using glucose as a carbon source.

Figure 1. Effect of carbon sources on DM and DP efficiency. 12

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3.4 Optimal conditions for demineralization using Lactobacillus rhamnoides fermentation

During fermentation, accumulated organic acid reacts with the calcium carbonate in the shrimp shells to produce calcium lactate, which can be removed by washing [20]. Therefore, the quantities of acid may be

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responsible for the degree of demineralization of shrimp shells. A series

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of single factor conditions were optimized to improve the acid production

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capacity and the DM efficiency of Lactobacillus rhamnoides, and the

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conditions included inoculum level, initial pH, cultured temperature,

lP

glucose concentration, and cultured time. When the inoculum level varied from 2% to 12%, the changes in pH, ash, and DM efficiency were

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determined. When the inoculated level was 4%, the pH of the

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fermentation broth was 4.23, and then the pH value did not decrease with the increase of the inoculated level. Under this inoculated level, the maximum DM efficiency (85.8%) was obtained. Similarly, the initial pH varied from 5 to 7.5, when the initial pH was 6.5, the pH of the fermentation broth was 3.98, and the maximum DM efficiency arrived 86.6%. Whereafter, the cultured temperature and glucose concentration were studied，as can be seen from Figure 2, the cultured temperature had a great influence on DM efficiency，which led to a significant difference (P < 0.05 ) in the removal rate of calcium carbonate. When the cultured temperature was 37 ℃, the efficiency of DM reached the maximum 13

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point at 95.2%, so the optimum temperature was 37 ℃. When glucose concentration reached 5%, the DM efficiency was not increased with glucose concentration. Based on the above-optimized conditions, the pH, ash and DM efficiency were determined at different cultured time from 0 to 72 h, the DM efficiency increased with prolonged cultured time, but

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reached equilibrium at 48 h with DM efficiency of 97.5%. We finally determined the cultured conditions (inoculum level 4%, initial pH 6.5,

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cultured temperature 37 ℃, glucose concentration 5%, cultured time 48

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h) of Lactobacillus rhamnoides. Under these conditions, the DM

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in the final residue was 1.2%.

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efficiency was 97.5%, the pH of fermentation broth was 3.4, and the ash

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Figure 2. Changes in the pH ( ), ash ( ) and DM ( ) efficiency of fermentation supernatants from Lactobacillus rhamnoides under different conditions. Bars with different letters are significantly different (P < 0.05).

3.5 Quantification of organic acids As showen in Table 2, the fermentation broth of Lactobacillus rhamnoides was rich in organic acids. Succinic, propionic, acetic, lactic, malic, formic acid were measured in the cultured supernatant at a total amount of 50357.3 mg/kg when the DM efficiency was 97.5%. The total 15

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organic acids amount and the DM efficiency were much higher than some studies, such as the case of Liu et al. [21]，who found that the total organic acids concentration in co-fermented liquid was 17889.9 mg/l, and the DM efficiency was 93.5%. Mao et al. [22] reported the total amount of organic acids in the cultured supernatant was 5426.74 mg/l, but the

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DM efficiency was not mentioned.

Quantification of organic acids of FBLA

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Table 2

succinic acid

propionic acid

acetic acid

lactic acid

malic acid

formic acid

FBLA (mg/kg)

nd

nd

4331.9

8585.8

36239.0

1200.6

nd

Total contents (mg/kg)

50357.3

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citric acid

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FBLA: fermentation broth of Lactobacillus rhamnoides.

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n.d.: below detection limit;

3.6 Optimal conditions for deproteinization using BA01 fermentation A series of single factor conditions were optimized to improve the protease activity and the DP efficiency of BA01. These single factor conditions were the same as above, and the results were shown in Figure 3. When the inoculum level was 6%, the DP efficiency was highest. The influence of initial pH was studied and the optimum initial pH of the protease activity was 6.5. The investigation of cultured temperature on protease activity displayed maximum activity at 37 ℃, the protease 16

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activity was 593.4 U/mL, and the DP efficiency was 89.7%. Afterward, the glucose concentration was studied, the DP efficiency increased as the concentration of glucose increased, and reached the highest value (95.5%) when the glucose concentration was 4%, thereafter, the DP efficiency did not increase with the increase of the glucose concentration, so the

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optimum concentration of glucose was 4%. Eventually, the protease activity and the DP efficiency were monitored over the cultured time

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range 12-108 h to determine the cultured time profile. The protease

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activity and the DP efficiency increased with cultured time, but they

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reached a steady level at 84 h with protease activity of 701.3 U/mL and

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DP efficiency of 96.8%. We finally determined the cultured conditions

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(inoculum level 6%, initial pH 6.5, cultured temperature 37 ℃ glucose concentration 4%, and cultured time 84 h) of BA01. Under these

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conditions, the DP efficiency was 96.8%, the protein in the final residue was under 1.5%, the protease activity of fermentation broth was 701.3 U/mL, and chitin yield was 19.6%.

17

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) efficiency of fermentation supernatants from BA01 under

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DP (

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Figure 3. Changes in the protease activity ( ), protein contents ( ) and

different conditions. Bars with different letters are significantly different (P < 0.05).

3.7 Fourier transform infrared (FT-IR) spectra analysis FT-IR spectra of samples were shown in Figure 4. The CTF (chitin prepared from successive two-step fermentation) showed a characteristic peak at 1652 and 1620 cm-1 which were corresponding to the amide I region and a signal at 1554cm-1 corresponding to the amide II region. The spectrum of CTF and CC (commercial chitin) was lacking the absorbance 18

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peak at 1540 cm-1 when CTL had the absorbance peak at 1540 cm-1, where proteins would normally give rise to absorption [23]. Absorbance peak at 3427 and 3255 cm-1 was recognized as O–H and N-H stretching vibration, respectively [24]. A medium intensity peak was observed at 3102 cm-1, which was corresponding to characteristic of amide II and

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α-chitin, being either an overtone of amide I or result of Fermi resonance between the overtone of amide II band and N-H stretching band [25].

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Furthermore, the other peaks characteristic of chitin at around 1375 (C–

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H), and 950–1200 cm-1 (C–O–C and C–O) were observed in CTF. Those

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results were similar to CC. The other peaks characteristic of the carbonate

lP

group at 1440, 874, and 725 cm-1 were not observed in CTF and CC

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due to the absence of calcium carbonate [26].

Figure 4: FT-IR spectra of CTL: chitin fermented by Lactobacillus 19

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rhamnoides (A); CTF: chitin prepared from successive two-step

fermentation (B); CC: commercial chitin (C). 3.8 Solid-state 13C CP/MAS NMR spectra analysis The Solid-state

13

C CP/MAS-NMR spectra of samples were shown in

Figure 5. The spectrum of 105.01ppm (C1), 84.14 ppm (C4), 76.64 ppm

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(C5), 74.41 ppm (C3), 61.95 ppm (C6), 56.14 ppm (C2), and 23.79 ppm

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(C8) were detected in commercial chitin and CTF, indicating high

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structural homogeneity. Besides, the 13C signals for C5 (76.64 ppm) and

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C3 (74.41 ppm) were separated into two signals clearly. These were

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similar to the commercial α-chitin. On the contrary, the β-chitin from squid pens, the C3 and C5 merge into a single peak at 75.0 ppm [27].

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Moreover, the peak of the protein in the 20-40 ppm region was observed

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in CTL, but not in CTF and CC, which demonstrated that there were no protein in CTF.

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Figure 5: Solid-state 13C CP/MAS NMR spectra of CTL: chitin fermented by Lactobacillus rhamnoides (A); CTF: chitin prepared from successive two-step fermentation (B); CC: commercial chitin (C). 3.9 X-ray diffraction patterns (XRD) X-ray diffraction patterns (XRD) were shown in Figure 6. Chitin is a

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semicrystalline polysaccharide polymer, which can be seen in the XRD

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patterns. In this study, CTF was compared with CC, the reflection

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crystalline peaks at 9.35◦, 12.75◦ and 19.47◦ in CTF was identified as

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diffraction planes (0 2 0)，(0 2 1) and (1 1 0) of the crystal structure,

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respectively. Crystalline peaks of CTF nearly overlapped with the

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polymorph.

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reference CC samples, which showed that CTF corresponded to the

Figure 6: X-ray diffraction patterns (XRD) of CTL: chitin fermented by Lactobacillus rhamnoides (A); CTF: chitin prepared from successive 21

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two-step fermentation (B); CC: commercial chitin (C). 3.10 Thermogravimetric analysis The TG/DTG analysis results showed that the mass loss of CTF: chitin prepared

from

successive

two-step

fermentation

(A);

CC:

commercial chitin (B). The mass loss of CTF (A) was 5% in the first step

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caused by water evaporation in the chitin structure and 68% in the second

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step due to polymer degradation, respectively [28]. The maximum

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thermal degradation temperature was 385 ℃

for CTF (A) and

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commercial chitin. The maximum thermal degradation value of α-chitin

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differed between 350 and 400 ℃ which has been reported by previous studies [29-31]. CTF (A) and commercial chitin showed a similar trend.

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Furthermore, the thermal stabilities of CTF were close to the thermal

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stability of the commercial chitin.

Figure 7: Thermogravimetric analysis (TGA) of CTF: chitin prepared from successive two-step fermentation (A); CC: commercial chitin (B). 22

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3.11 Scanning electron microscope (SEM) The scanning electron microscope (SEM) of samples were shown in Figure 8. It can be seen distinct microstructures of the four samples. SSP (shrimp shells powders) showed that too many impurities structures were tightly bound to chitin, which included protein and calcium carbonate.

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The microfibrillar structure cannot be observed because the surface was

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too rough. When the SSP were removed from the calcium carbonate and

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become CTL (chitin fermented by Lactobacillus rhamnoides), the surface

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morphology has changed significantly, but because the surface of the

lP

chitin was still covered with protein, its microfibrillar structure was not apparent. CTF (chitin prepared from successive two-step fermentation)

the organization

of α–chitin

extracted

from shrimp

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following

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showed smooth surface and obvious fractured structure which was

(Litopenaeus vannamei), Antarctic krill (Euphausia superba) [21, 32]. It’s worth noting that CTF showed a more noticeable microfibrillar crystalline structure than CC in SEM, CC did not have apparent microfibrillar structure but showed the densely fractured structure and the sign of perforations.

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Figure 8: Scanning electron microscopic examination of SSP: shrimp

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shells powders (A); CTL: chitin fermented by Lactobacillus rhamnoides

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(B); CTF: chitin prepared from successive two-step fermentation (C); CC:

commercial chitin (D)

3.12 Quantification of total amino acids The type and amount of amino acids have a great impact on the nutritional value of food. Table 3 showed the type and amount of the amino acids of shrimp shells, fermentation broth of Lactobacillus rhamnoides and fermentation broth of BA01, and the total content of amino acids in shrimp shells, the fermentation broth of Lactobacillus rhamnoides and the fermentation broth of BA01 was 349.12 mg/kg, 22.90 24

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mg/kg, 367.66 mg/kg, respectively. These results were similar to those reported by Bueno-Sola et al. [33]. The low amount of amino acids in the fermentation broth of Lactobacillus rhamnoides was probably due to the strain's inability to produce proteases to hydrolyze proteins. It is worth mentioning that tryptophan was not determined as it was destroyed during

of

acid hydrolysis [22]. Phenylalanine was the amino acid present in superior quantity at 25.12 mg/kg, and methionine had the lowest

ro

concentration of 3.72 mg/kg, while threonine was the dominant amino

-p

acid at 63.12 mg/kg and tyrosine had a limiting concentration of 0.73

re

mg/kg in the fermentation broth of BA01. The total essential amino acid

lP

content in shrimp shells, fermentation broth of Lactobacillus rhamnoides

respectively.

na

and fermentation broth of BA01 was 37.72%, 34.15%, 44.49%,

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Essential amino acids (EAA) are those cannot be synthesized or synthesized at a rate that does not meet the needs of living organisms, which must be taken from food to meet basic needs [34]. In fermentation broth of BA01, essential amino acids accounted for 44.49% of total α-amino acids (TAA). The composition of fermented broth of BA01 (EAA/TAA was 44.49%) was comparable to that of the FAO/WHO [35] requirement pattern (EAA/TAA was about 0.40). These results indicated that the fermented broth of BA01 possesses high nutritional value and can be considered used as an ingredient in animal and plant diets, even food. 25

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Table 3 The amino acid composition of Shrimp shells powders and fermentation broth of Lactobacillus rhamnoides and BA01

SSP

FBLA

FBBA

Asp

37.12

2.03

34.71

Thr

16.13

0.71

63.12

Ser

22.72

1.12

Glu

58.31

3.51

Gly

25.31

Ala

25.41

Cys

7.41

of

Amino acid

34.13

2.64

29.32

0.74

12.72

24.72

1.31

23.61

3.72

0.52

2.71

16.51

0.72

15.52

24.21

1.32

25.91

Tyr

20.51

0.71

0.73

Phe

25.12

1.33

11.61

His

20.61

0.82

23.32

Lys

21.31

1.91

21.11

Amino acid total

349.12

22.90

367.66

EAA/TAA

37.72

34.15

44.49

lP

3.51

re

53.51

na

-p

ro

15.63

Val Met Ile

Jo ur

Leu

SSP: shrimp shells powders FBLA: fermentation broth of Lactobacillus rhamnoides. FBBA: fermentation broth of BA01 26

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4. Conclusion In this study, the chitin obtained by successive two-step fermentation preserved the special physicochemical and structural properties of chitin. The final DM was 97.5%, ash content was 1.2%, DP was 96.8%, and protein content of 1.5%. The fermentation broth of Lactobacillus

acid

(36239.0

mg/kg),

which

is

the

of

rhamnoides was rich in organic acid (50357.3 mg/kg), especially lactic

main

reason

for

good

ro

demineralization effect. The protease activity of BA01 fermentation broth

-p

was 701.3 U/mL, higher enzyme activity was the main reason for good

re

protein removal. These two strains have good growth adaptability during

lP

the fermentation of shrimp shells and do not need any other ingredients

na

except glucose. Therefore, it is feasible to prepare chitin by microbial fermentation. Moreover, in order to make full use of shrimp and crab

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shell resources, the amino acid composition of fermentation broth was detected. All in all, compared with hazardous chemical methods, the microbial fermentation is a relatively simple and environmentally friendly method. 5. Acknowledgements This study was supported by the National Key R&D Program of China (2018YFC0311305), the Key Research and Development Program of Shandong Province (2019GHY112015, 2017YYSP018).

27

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6. References [1] S. Islam, Md, KHAN, Saleha, TANAKA, Masaru, Waste loading in shrimp and fish processing effluents: potential source of hazards to the coastal and nearshore environments, Marine Pollution Bulletin 49(1) (2004) 103-110.

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[22] X. Mao, P. Liu, S. He, J. Xie, F. Kan, C. Yu, Z. Li, C. Xue, H. Lin, Antioxidant Properties of Bio-active Substances from Shrimp Head Fermented by Bacillus licheniformis OPL-007, Applied Biochemistry and Biotechnology 171(5) (2013) 1240-1252. [23] J.D. Goodrich, W.T. Winter, alpha-Chitin nanocrystals prepared from shrimp 31

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Highlights 1. Chitin was extracted from shrimp shells powders by successive two-step fermentation. 2.

The best microorganisms were Lactobacillus rhamnoides and Bacillus amyloliquefaciens.

3. The DM efficiency was 97.5% and the DP efficiency was 96.8%.

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na

lP

ro

re

-p

with those of commercial products

of

4. The characterization of chitin obtained by fermentation are consistent

34