Team:IIT Kharagpur/project

Causes of food spoilage

Food spoilage can be defined as rendering of a product undesirable for consumption meanwhile causing serious economic loss of the owners. Food spoilage can be attributed to both inorganic and organic forces. These include light, heat, humidity, biochemical reactions and microbial actions most commonly by microbes such as bacteria, yeast and moulds. Microbial food spoilage manifests itself as visible growth and food textural changes (Gram and others 2002).The off-taste and off-odor due to release of metabolites by microbes results in rejection of the food. Microorganisms produce saccharolytic, proteolytic, pectinolytic, and lipolytic enzymes whose metabolic end products are associated with food spoilage (Braun and others 1999; Loureiro 2000; Ragaert and others 2007).

Quorum Sensing

Bacteria use signaling molecules for inter- and intracellular communication. This phenomenon of bacterial cell-to-cell communication is known as quorum sensing controlling a broad range of activities. The modulation of gene expression in bacteria by quorum sensing results in phenotypic changes leading to their better adjustment to environmental conditions during growth . Recent explosion in studies of such complex chemically coordinated system has made clear that the ability to communicate both within and between species is critical for bacterial survival and interactions in natural habitat. It can be said bacteria takes decisive action based upon assessing local cell densities.

Cell-to-cell communication depends on the production of, secretion of, and response to small, diffusible signal molecules called "autoinducers". The signal molecules are produced and secreted at a basal level during bacterial growth. Their concentration in the environmental medium or matrix increases as the bacterial population expands, and when it reaches a threshold level (quorum level). It induces phenotypic effects by regulating quorum-sensing dependent target gene expression . This phenomenon occurs without any external intervention and is also referred to as auto induction

Quorum Sensing in Food Spoilage

Quorum sensing or cell-to- cell communication is employed by a diverse group of bacteria including those commonly associated with food to communicate with each other by producing the signaling molecules, autoinducers. It has been that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes. Several proteolytic, lipolytic, chitinolytic, and pectinolytic activities associated with the deterioration of foods are regulated by quorum sensing. Moreover, several types of signaling molecules (like AHL) have been detected in different spoiled food products. Milk and dairy products are easily susceptible to spoilage by psychrotrophic bacteria such as pseudomonads. These Gram-negative bacteria produce extracellular proteinases, lipases, lecithinases, and glycosidases responsible for food spoilage. Quorum sensing is involved in regulation and expression of these biochemical substances was elucidated by a study in Serratia proteamaculans strain B5a. The inoculation of pasteurized milk with wild-type S.proteamaculans caused spoilage after 18 h of storage at room temperature, while the inoculation with a mutant having an inactivated sprI (inducer (HSL) producing gene) gene did not result in spoilage. However, the addition of 3-oxo-C6-HSL to milk inoculated with the sprI mutant caused its spoilage, implying the role of signaling molecules in the spoilage of milk.

Food spoilage bacteria can produce AHLs even when they are present at a lower concentration in the food substrate. The AHLs (mainly 3-oxo-C6-HSL) have been produced by the inoculated (under conditions that simulated food environments of 5 ◦C, reduced oxygen, and 4% NaCl) and indigenous members of the Enterobacteriaceae at lower concentrations of 106 CFU/mL and 105 to 106 CFU/g, respectively

The pectinolytic activity of Pseudomonadaceae or Enterobacteriaceae (mostly Erwinia spp.) growing to high-cell densities (108 to 109 CFU g/L) in fruits and vegetables causes enzymatic browning, off-tastes, off-odors, and/or texture breakdown resulting in their spoilage (Liao 1989). Erwinia and Pseudomonas produce various pectinolytic enzymes, namely, pectin lyases, pectate lyase, polygalacturonase, and pectin methyl esterases, which are responsible for the spoilage of ready-to-eat vegetables, also produce a broad range of AHLs (mainly 3-oxo-C6-HSL and C6-HSL) (Rasch and others 2005).

Table: Bacterial food spoilage influenced by quorum-sensing-regulated phenotypes.
Organism	Food product	Signal-dependent phenotype	Signaling molecules	References
Pseudomonas fluorescens 395	Milk	Proteolytic milk spoilage	C4-HSL and 3OC8-HSL	Liu and others (2007)
Serratia proteomaculans strainB5a	Milk	Lipolytic and proteolytic milk spoilage	3-oxo-C6- HSL	Christensen and others (2003)
Pseudomonas fluorescens	Milk	Proteolytic milk spoilage	L-HSL α-amino-γ -butyrolactones	Dunstall and others(2005)
Pseudomonas phosphoreum and Aeromonas spp	Cod fillets	Chitinolytic activity	3-hydroxy-C8-HSL	Flodgaard and others(2005)
Erwinia carotovora	Vegetables	Cellulolytic and proteolytic Spoilage	3-oxo-C6- HSL	Jones and others (1993)
Pectobacterium sp. A2JM	Bean sprouts	Pectinolytic and proteolytic Spoilage	3-oxo-C6- HSL	Rasch and others (2005
Serratia plymuthica RVH1	Vegetables	Chitinase and protease Activity	3-oxo-C6- HSL and C6-HSL	Van Houdt and others (2007)
Hafnia alvei and Serratia spp.	Vaccum packed Meat	Proteolytic spoilage	N-3-oxohexanoyl HSL	Bruhn and others (2004)
Pseudomonas spp.	Meat	Biofilm formation and proteolytic spoilage	AHLs	Jay and others (2003)
Photobacterium phosphoreum and Aeromonas spp.	Cod fillets	Chitinolytic spoilage	3-hydroxy-C8-HSL	Flodgaard and others (2005)

Mechanism (the LuxR/LuxI system)

The typical LuxR/LuxI system was first discovered in Vibrio fischeri as the first quorum sensing circuit. There are five luciferase structural genes (luxCDABE ) and two regulatory genes (luxR and luxI ) required for quorum sensing–controlled light emission in V. fischeri. The genes are arranged in two adjacent but divergently transcribed units. luxR is transcribed to the left, and the luxICDABE operon is transcribed to the right. The LuxI protein (square) is responsible for synthesis of the HSL autoinducer N-(3-oxohexanoyl)-homoserine lactone (hexagons). As the cell-population density increases, the concentration of the autoinducer increases both intra- and extracellularly. At a critical autoinducer concentration ((1-10 ug/ml), the LuxR protein (circle) binds the autoinducer. The LuxR-autoinducer complex binds at the luxICDABE promoter and activates transcription of this operon. This action results in an exponential increase in autoinducer synthesis via the increase in transcription of luxI and an exponential increase in light production via the increase in transcription of luxCDABE. The LuxR-autoinducer complex also binds at the luxR promoter, but in this case the complex represses the transcription of luxR. This negative action compensates for the positive action at the luxICDABE promoter. The oval represents a bacterial cell

Our Project

So ever got sick of the food poisoning more by doctor’s heavy fee and prescription tabloid than the actual pathological state of affairs. To add fire to fury is the simple mind nibbling thought that this could have been easily detected. And! Everything was fine, you checked the outdated milk stuffed behind the large cereal bowls by – sniffing it! Or Tasting it! Or just fundamental theorem of projecting last time’s experience over two more days! Old kitchen’s wives tales are not always helpful are they?”But what alternative do we have”, you say, – quick and efficient, while not being too expensive. That is why you need to pay attention to what follows! iGEM IIT Kharagpur targeted the ancient cause of microbial animosity among mankind i.e. microbial spoilage of food and subsequent pathological disorder which follows. Out of the long list of possible candidates we chose to detect bacteria which releases AHL( Acyl Homoserine Lactone) molecules as a quorum signal which gives a qualitative idea of the microbial population in the system. As explained in project background, quorum signal molecules regulate expression of certain genes (different in every organism) which can produce light illuminating peptides in Vibrio fischerie. We decided to develop a modified strain from purchased E.coli DH5-alpha strain. The modification includes presence of a singular plasmid which consists of luxR gene put under a constitutive promoter, a luxpR sequence which is a luxR-AHL complex inducible promoter site. Our idea is based on around the premise that AHL is constitutively produced by bacteria and as their population grows the AHL concentration grows. Since AHL is freely diffusible through cell membrane its intracellular concentration is equal to extracellular concentration. In presence of luxR protein to which it has a binding affinity, post binding it forms an activating complex which activates the gene downstream of luxPR sequence. The gene which we have introduced is CrtEBI which is a cluster of three protein producing region, proteins being CrtE, CrtB, CrtI. E.coli naturally synthesis IPP and DMAPP via non mevalonate pathway, which are readily converted to FPP (farnesyl pyrophosphate). FPP is sequentially converted by CrtE, CrtB, CrtI to red lycopene. It is a colored pigment which is red in color. This red signal should be considered as red alert against the food spoilage measurement. Our wet lab team decided to make use of three biobrick assemblies ordered ( as mentioned above) from iGEM part repository. The final biobrick planned was to have parts from all three plasmids ( 2 genes and 1 promoter).