GC-MS-Based Metabolomics Analysis of Prawn Shell Waste Co-Fermentation by Lactobacillus plantarum and Bacillus subtilis

: GC-MS-based metabolomics were used to investigate metabolic changes in prawn shell waste during fermentation. Microbial strains Lactobacillus plantarum and Bacillus subtilis were co-fermented in a shake ﬂask comprising of 5% ( w / v ) prawn shell waste and 20% ( w / v ) glucose as a carbon source. Analysis of the prawn shell waste fermentation showed a total of 376 metabolites detected in the culture supernatant, including 14 amino acids, 106 organic acids, and 90 antimicrobial molecules. Results show that the liquid fraction of the co-fermentation is promising for harvesting valuable metabolites for probiotics application. Conceptualization, W.N.C.; methodology, Y.N.T. and J.H.Z.; software, Y.N.T. and J.H.Z.; validation, Y.N.T. and J.H.Z.; formal analysis, Y.N.T. and J.H.Z.; investigation, Y.N.T. and J.H.Z.; resources, W.N.C.; data curation, Y.N.T. and J.H.Z.; writing—original preparation, Y.N.T.; writing—review and editing, Y.N.T.; visualization, W.N.C.; W.N.C.; project administration, W.N.C.; W.N.C.


Introduction
The industrial seafood processing industry generates more than 1 million metric tons of dry weight of shellfish waste annually [1]. As the heads and exoskeletons of shellfish that comprise about 50-60% of their total weight are not suitable for human consumption, these shellfish residues are discarded as seafood processing waste by ocean dumping, incineration, or disposal in landfills [2]. This has contributed to both land and sea pollution, hence sparking scientific and environmental interest to develop techniques to recover and utilize the biopolymers in shellfish waste [3].
Prawn shell waste is chemically composed of 20-30% chitin, 20-40% protein, 30-60% minerals, and 0-14% lipids [4]. Currently, crustacean waste serves as the largest source of chitin or its deacetylated derivative chitosan [5]. Chitin, a polysaccharide with a similar structure to cellulose, is an N-acetyl-glucosamine biopolymer with α-1,4 bonds between each monomeric unit [6]. The isolation of chitin involves deproteinization, demineralization, and bleaching [7]. Traditional chemical methods involve the use of highly concentrated sodium hydroxide to carry out deproteinization and highly corrosive hydrochloric acid to carry out demineralization [8]. Other than the formation of toxic waste, undesired by-products such as irregularly deacetylated polymers result [9]. In addition, the protein and carotenoid components of the prawn shell waste are rendered useless [10].
Research has focused on using environmentally friendly processes such as biological co-fermentation by lactic acid bacteria and protease producing bacteria [11]. The lactic acid produced during fermentation reacts with the calcium carbonate in the prawn shell waste, leading to the formation of calcium lactate, which can be separated from the chitin fraction [12]. Proteolytic enzymatic action also simultaneously hydrolyzes the protein fraction of prawn shells to recover chitin [13]. Much attention has been directed at optimizing the extracellular production of the chitinase enzyme by the selection of appropriate micro-organisms [14]. Various factors such as glucose concentrations, inoculum sizes, pH, temperature, and length of fermentation influence the fermentation process as well as deproteinization and demineralization efficiencies [15].

Determination of Chitin Yield and Purity
The mass of the crude chitin obtained was weighed after being dried in the vacuum oven overnight to determine its yield. The Lowry's test for residual protein was carried out to ascertain the purity of the recovered chitin. Firstly, 5,10,15,20,25, and 30 µL of 2 mg/mL bovine serum albumin (BSA) was added to 195,190,185,180,175, and 170 µL of deionized water respectively to form a range of 200 µL protein standards for the construction of a protein calibration curve. Then, 1 mL of Lowry's solution was added to the protein standards and left to react for 15 min, after which 100 µL of 1 N Folin's Phenol reagent was added and the protein standards were left to react for another 30 min. Absorbance was measured at 750 nm and the values were plotted into a graph of absorbance versus µg protein.
Fifty milligrams of the extracted crude chitin was then treated with 10 mL of 1 M aqueous sodium hydroxide solution for 24 h at 70 • C. 1 mL of Lowry's solution and 100 µL of 1 N Folin's Phenol reagent was similarly added to the boiled NaOH supernatant to determine the residual protein content of the recovered chitin [39].

Metabolomics Analysis by GC-MS
A total of 376 metabolites were detected by GC-MS. Fourteen amino acids were detected in the fermentation, with the highest quantity being alanine (4642.67 mg/L), followed by proline (91.76  Full detailed results for the detected metabolites are shown in Tables 1-4 below.

Chitin Yield and Purity Calculations
From 5.0 g of prawn shell waste, 20 g of glucose, and 100 g of deionized water, the dry weight of crude extracted chitin was found to be 0.50 ± 0.01 g, translating to an overall fermentation yield of 0.50/125.0 × 100% = 0.4%.
Lowry's test was performed on 1 mL of supernatant extracted from 50 mg chitin heated in 10 mL NaOH and its absorbance was found to be 0.213, corresponding to 20 µg of protein when compared against the protein calibration curve (Figure 1). This translates to a residual protein of 200 µg per 50 mg chitin, which is a residual protein content of 200/50,000 × 100% = 0.4%.
Lowry's test was performed on 1 mL of supernatant extracted from 50 mg chitin heated in 10 mL NaOH and its absorbance was found to be 0.213, corresponding to 20 μg of protein when compared against the protein calibration curve (Figure 1). This translates to a residual protein of 200 μg per 50 mg chitin, which is a residual protein content of 200/50,000 × 100% = 0.4%.

Discussion
Bacteria species coexist with neighboring microorganisms in a dynamic community by producing small metabolites in response to environmental changes such as biotic and abiotic stresses. These volatile organic and inorganic compounds are released during interspecies bacteria interactions due to competition and cooperation, forming soluble metabolites in the supernatant [134]. Detection and quantification of these bacteria volatile compounds have always been of great interest in the food, cosmetic, flavor, and fragrance bioprocessing industry as well as in the clinical and medical field. However, analysis of bacteria volatile compounds has remained challenging due to the wide abundance of metabolites and the complexity of the culture medium from where they are extracted.
The co-fermentation of prawn shell waste and 20% glucose by Lactobacillus plantarum and Bacillus subtilis for chitin extraction produced bacteria volatile metabolites of various chemical classes. Fatty acid derivatives such as hydrocarbons, ketones and alcohols, organic acids, as well as sulphur and nitrogen-containing compounds were detected in the culture supernatant. These metabolites were generally produced by different catabolic pathways such as glycolysis, proteolysis, and lipolysis to break down the proteins, fats, and minerals residual in the prawn shell waste [135]. Linear-chained hydrocarbons detected were most probably derived from products of the fatty acid biosynthetic pathway. Both short-chain alkanes and longer-chain hydrocarbons were found in the culture supernatant, testifying to the ability of the microbial strains to synthesize branched hydrocarbons.
Methyl ketones detected were probably produced from the decarboxylation of fatty acids [136]. For example, 3-hydroxy-2-butanone (72.50 mg/L) or acetoin detected might have been derived from pyruvate fermentation. Long-chain aliphatic alcohols such as 1-decanol (1.75 mg/L) were probably produced through the oxidation of fatty acid derivatives. Significant production of butanediol (139.50 mg/L) was detected due to the presence of glucose as the main nutrient in the growth medium. Shortchain branched alcohols such as 3,3-dimethyl-1-butanol (18.60 mg/L) detected might have been produced from the enzymatic conversion of branched chain amino acids such as leucine.
Several short-chain fatty acids were detected in the culture supernatant such as acetic acid (71.94 mg/L), propanoic acid (1184.32 mg/L), and butanoic acid (4399.87 mg/L). These saturated aliphatic organics acids most probably resulted from bacteria fermentation of carbohydrates. Glyoxylic acid

Discussion
Bacteria species coexist with neighboring microorganisms in a dynamic community by producing small metabolites in response to environmental changes such as biotic and abiotic stresses. These volatile organic and inorganic compounds are released during interspecies bacteria interactions due to competition and cooperation, forming soluble metabolites in the supernatant [134]. Detection and quantification of these bacteria volatile compounds have always been of great interest in the food, cosmetic, flavor, and fragrance bioprocessing industry as well as in the clinical and medical field. However, analysis of bacteria volatile compounds has remained challenging due to the wide abundance of metabolites and the complexity of the culture medium from where they are extracted.
The co-fermentation of prawn shell waste and 20% glucose by Lactobacillus plantarum and Bacillus subtilis for chitin extraction produced bacteria volatile metabolites of various chemical classes. Fatty acid derivatives such as hydrocarbons, ketones and alcohols, organic acids, as well as sulphur and nitrogen-containing compounds were detected in the culture supernatant. These metabolites were generally produced by different catabolic pathways such as glycolysis, proteolysis, and lipolysis to break down the proteins, fats, and minerals residual in the prawn shell waste [135]. Linear-chained hydrocarbons detected were most probably derived from products of the fatty acid biosynthetic pathway. Both short-chain alkanes and longer-chain hydrocarbons were found in the culture supernatant, testifying to the ability of the microbial strains to synthesize branched hydrocarbons.
Methyl ketones detected were probably produced from the decarboxylation of fatty acids [136]. For example, 3-hydroxy-2-butanone (72.50 mg/L) or acetoin detected might have been derived from pyruvate fermentation. Long-chain aliphatic alcohols such as 1-decanol (1.75 mg/L) were probably produced through the oxidation of fatty acid derivatives. Significant production of butanediol (139.50 mg/L) was detected due to the presence of glucose as the main nutrient in the growth medium. Short-chain branched alcohols such as 3,3-dimethyl-1-butanol (18.60 mg/L) detected might have been produced from the enzymatic conversion of branched chain amino acids such as leucine.
Several short-chain fatty acids were detected in the culture supernatant such as acetic acid (71.94 mg/L), propanoic acid (1184.32 mg/L), and butanoic acid (4399.87 mg/L). These saturated aliphatic organics acids most probably resulted from bacteria fermentation of carbohydrates. Glyoxylic acid (4.68 mg/L) detected could either have been produced in the tricarboxylic acid cycle or generated during amino acid metabolism, for example during the degradation of glycine (25.56 mg/L), threonine (91.73 mg/L), and proline (91.76 mg/L). Indole (22.32 mg/L) biosynthesis, another by-product of amino acid catabolism, was also detected in the fermentation supernatant [137].
An oxidative deamination of many amino acids might have also led to the production of aldehydes, ketones, or alcohols detected. For example, the degradation of 1,4-dihydrophenyalanine (2.92 mg/L) might have served as the first step of aromatic volatile compounds synthesis, producing benzene, its carbohydrate derivatives, as well as other benzenoid volatiles. Many volatile organic compounds produced by Lactobacillus plantarum and Bacillus subtilis have been reported to display antimicrobial activity. Among these known antimicrobial metabolites, benzenoids are the most represented in quantity compared to alkanes, aldehydes, ketones, acids, and alcohols. While a huge majority of antimicrobial benzenoid volatiles have a benzene core linked to a fatty acids derivative, benzenoids are very diverse and can be linked with carbohydrate chains containing nitrogen and sulphur [138].
The antimicrobial mode of action of these bacteria volatile organic compounds might arise from their lipophilic nature, which enables them to destabilize the cell membrane integrity of antagonistic pathogens, inhibiting their growth [139]. Besides benzenoids, nitrogen-containing volatile organic compounds are another important group of antimicrobial metabolites, consisting of non-cyclic amides and amines as well as cyclic azoles, pyrazines, pyridazines, and pyrimidines. Pyrazine (33.19 mg/L), pyridazine (3.29 mg/L), and pyrrolopyrimidine (3.33 mg/L) were detected in the Lactobacillus plantarum and Bacillus subtilis co-fermentation supernatant. Pyrazine, which is the most strongly represented in antimicrobial activity among them, is either formed from the non-enzymatic animation of acyloins or derived from aminoketone intermediates produced from amino acid catabolism. This testifies to the successful breakdown of amino acids from the prawn shell waste.
Antimicrobial active metabolites may have potential use as natural preservatives to control the growth and inactivate undesired microorganisms in food [140]. For example, lactic acid (1055.39 mg/L) and acetic acid (71.94 mg/L) are produced by Lactobacillus plantarum in probiotics to compete for nutrients with other foodborne pathogens. Other organic acids such as propanoic acid (1184.32 mg/L) and butanoic acid (4399.87 mg/L) are also produced, which further reduce the pH of the culture medium. The production of other substances such as ethanol (31.01 mg/L), fatty acids such as 3-hydroxybutyric acid (18.19 mg/L), 3-hydroxysebacic acid (4.09 mg/L), and 3-hydroxpyruvic acid (1.32 mg/L), as well as 3-hydroxy-2-butanone (72.5 mg/L) further intensify its antimicrobial activity. The metabolomics results show that Lactobacillus plantarum is more heterofermentative than homofermentative as a variety of metabolites are generated from the degradation of hexoses.

Conclusions
Many useful metabolites are produced when Lactobacillus plantarum and Bacillus subtilis are fermented with prawn shell waste together with 20% glucose as a carbon source. Besides lactic acid, a variety of organic acids such as fatty acids and amino acids as well as several antimicrobial molecules were detected in the culture supernatant. This shows that protease-mediated protein hydrolysis of the prawn shells is successful in removing proteins, minerals, and fats from the prawn shells. While harnessing the solid fraction of the fermentation as chitin, the nutrient-rich liquid fraction may be used for probiotics applications.