Improved production of chitin from shrimp waste by fermentation with epiphytic lactic acid bacteria

Abstract

The epiphytic Lactobacillus acidophilus SW01 isolated from shrimp waste (SW) was used in SW fermentation. During the fermentation the lag phase of SW01 was hardly observed. The pH of the fermentation broth decreased to 3.86 within 12 h and reached the lowest point at 3.73 after 48 h. This indicates a quick and deep acidification process by SW01. Besides, SW01 was observed to have high protease activity. As a result, the minerals and protein in SW were quickly removed with their contents decreasing to 0.73% and 7.8% respectively after 48 h fermentation. In the pilot scale fermentation, the pH was 3.99 and 3.86 respectively after 12 and 24 h fermentation. The mineral and protein contents were 0.98% and 8.44% respectively after 48 h fermentation. The residue of the fermented SW contains less than 1% minerals and can be easily transformed into chitin by a mere bleaching treatment.

Introduction

Chitin, the second largest carbohydrate source in nature, is widely distributed as a structural component of exoskeletons of crustaceans, insects and other arthropods, as well as a component of the cell walls of most fungi and some algae. It is an insoluble linear homopolymer of β-(1→4)-linked-N-acetyl-d-glucosamine (GlcNAc). The repeating structural unit of chitin is the dimer of GlcNAc (Muzzarelli et al., 2012). Chitin and its derivatives, such as chitosan and chito-oligosaccharides, have many important applications in the fields of pharmaceutical, food, agriculture, bioengineering and cosmetics and other related fields. Therefore chitin has attracted the attention of people for decades.

Nowadays, a widely used chitin source is shrimp waste (SW), which mainly consists of cephalothorax and exoskeleton and accounts for ca. 40% of the shrimp weight. The output of SW is very high worldwide. For example in China over 500 kilotons are produced annually. Chitin might be more profitably recovered from SW if an effective technology is developed.

In SW, chitin is linked with proteins and forms chitin-protein complex. Interspersed with the complex are minerals and a small amount of lipids and carotenoid. Traditionally, chitin is extracted from SW using strong acid and alkali to remove minerals, protein and lipids. This method has some disadvantages. For one, it causes part hydrolysis of chitin (Brine and Austin, 1981, Healy et al., 1994). Secondly, it produces a considerable amount of alkali waste water, which contains a lot of protein, lipids and carotenoid. In some cases the waste water is discharged without any treatment, which leads to serious environmental pollution. Furthermore, the protein, lipids and carotenoid cannot be recovered. Even though nowadays the waste water can be treated by couple-membrane filtration and the protein and sodium hydroxide can be recovered (Zhao, Xia, and Zhao, 2011), the capital and operating costs for such treatment are very high. A newer approach in chitin production is the use of proteolytic microorganisms (Jo et al., 2008) or protease (Ghorbel-Bellaaj et al., 2011) to remove the protein in SW. However, this approach leaves the minerals intact. As a result, strong acid has to be used to remove the minerals to produce chitin.

To solve the above problems, some researchers have tried to use lactic acid bacteria (LAB) to ferment SW to extract chitin. Under this method, lactic acid removes most minerals from SW. And most protein is hydrolyzed by protease and transferred into the fermentation broth. Chitin, protein, and carotenoid in SW can be obtained simultaneously. Moreover, the above-mentioned environmental pollution is avoided. Besides, the chitin obtained in this way has a higher molecular weight and crystallinity than the chemically extracted chitin (Pacheco et al., 2011).

This method is very promising. As a result, it has been developed, improved and optimized by different researchers. For example, Zakaria, Hall, and Shama (1998) studied lactic acid fermentation of scampi waste in a rotating horizontal bioreactor for chitin recovery. Shiraia et al. (2001) studied the effect of glucose concentration and inoculation level in lactic acid fermentation of SW for chitin recovery and SW preservation. Cira, Huerta, Hall, and Shirai (2002) studied the lactic acid fermentation of SW in pilot scale for chitin recovery. Bhaskar, Suresh, Sakhare, and Sachindra (2007), Sachindra, Bhaskar, Siddegowda, Sathisha, and Suresh (2007) and Choorit, Patthanamanee, and Manurakchinakorn (2008) separately optimized the lactic acid fermentation process for chitin and carotenoid recovery from SW. Pacheco et al. (2009) studied the effect of temperature in lactic acid fermentation of SW on chitin and carotenoid recoveries. Duan, Zhang, Lu, Cao, and Chen (2011) studied the fermentation of SW with 3 strains of symbiotic LABs to recover chitin and protein.

However, a problem common to all the above-mentioned researches is that the removal of minerals is insufficient. As a result, the fermented SW has to be treated with hydrochloride acid for further mineral removal. An important reason for this is the insufficient acidification in fermentation.

To improve the acidification in SW fermentation and promote the removal of minerals, our group has attempted different approaches. One way was to isolate epiphytic LABs from SW and use it in SW fermentation. This method was based on the assumption that epiphytic LABs were likely to grow better than others in SW and produce more acid. As expected, 3 strains of epiphytic LABs were isolated form SW. When growing in SW, one of them was found capable of producing much more acid than the other two. It was identified as L. acidophilus and named as SW01. The factors influencing its acid production and protein hydrolysis in SW fermentation was explored (Li, Zhuang, Duan, Liu, & Lin, 2011).

In this research, SW01 was further compared with other LABs in terms of acid production and protein hydrolysis in SW fermentation. The ingredient changes in SW fermentation with SW01 were investigated. Finally pilot scale fermentation of SW with SW01 was conducted.