Enzymes as Analytical Tools for the Assessment of Food Quality and Food Safety

C H A P T E R

10 Enzymes as Analytical Tools for the Assessment of Food Quality and Food Safety Kamaljit Kaur, Pragati Kaushal Department of Food Science and Technology, Punjab Agricultural University, Ludhiana, India

10.1 INTRODUCTION According to food and regulatory agencies, food quality and safety are the critical parameters common to all food items, and these parameters are of great economic importance. Evaluation of properties and composition of raw materials and food products are necessary to maintain food safety and quality because of the growing interest of the food industry and consumers in these concepts. Food safety relies on the contents of toxic compounds, hazardous microorganisms, toxins released by microorganisms, naturally present toxic substances, pesticides, and antinutritional compounds [1,2]. The quality control of food and confirmation of safety is an essential part of food analytical procedures. There are multiple customary analytical procedures such as GC, HPLC, and so forth for analysis of food safety and quality. However, the major drawback of these methods is complicated sample preparation procedures. Ongoing analytical procedures in the food industry are time consuming and depend on skilled labor, and these techniques are based on long separation methods, expensive instruments, and highly pure chemicals. In the field of fast screening, there is a need for complementary techniques to detect food quality and safety issues. Compared with conventional physical and chemical methods, enzymatic analysis is more specific and sensitive [3]. Blanching (treating vegetables with steam or boiling water for a short time) is an important unit operation for almost all vegetables that are to be canned, dried, or frozen. This process stops enzymatic activity that can cause loss of color, texture, and flavor. Blanching helps in other food preservation operations, as this unit operation reduces the multiple contaminating microorganisms

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on the surface of food. It softens the tissues of vegetable to facilitate filling into cans and removes air from intracellular voids. This unit operation is especially important in heat sterilization, as the time-temperature combination is designed to achieve a specified reduction in microorganisms and enzymes. A lot of research has been carried out for the design optimization and operational parameters of blanching processes for different vegetables. Lipoxygenase has been recommended for determining the storage stability of frozen vegetables [4]. As a part of analytical procedures, enzymes play a great role in food, environmental, pharmaceutical, and industrial analysis. Enzymes are usually considered standard analytical devices for determining the concentration of compounds that act as activators, substrates, or inhibitors of particular enzymes. The activity and concentration of selected enzymes is useful for evaluation of physically or nutritionally induced modifications in determining quality parameters of foods. Biosensors are some of the most suitable analytical methods for quality control and detecting contamination. This analytical method monitors the whole food chain process in a very systematic and cost-effective manner. Biosensors utilize enzymes to indicate the amount of a biomaterial. The key elements of biosensors include: bioreceptor, transducer, and a signal processing system. A bioreceptor generally consists of an immobilized biocomponent that detects the specific target analyte. Basic characteristics of biosensors include: linearity, sensitivity, selectivity, and response time. Biosensors are used in a number of applications in the area of food quality and safety [5]. ELISA (enzyme-linked immunosorbent assay) is a highly sensitive solid-phase enzyme immunoassay that employs an enzyme-linked antigen or antibody as a marker for the detection of specific antibodies, proteins, hormones, or peptides. It is used as a diagnostic tool in plant pathology, medicine, and particularly in food industries to check and maintain quality. In ELISA, an antigen must be immobilized on a solid surface, and then complexed with an antibody that is linked to an enzyme. A colored reaction product is obtained when an enzyme conjugated with an antibody reacts with a colorless substrate. The ELISA reactants, when immobilized to the microplate surface, result in separating the bound from unbound material during assay reactants at a fast rate. This property of ELISA to segregate nonspecifically bound materials makes it a powerful analytical tool for determination of specific analytes within a crude preparation. ELISA plays a great role in areas such as food analysis and safety, as it is widely used for detection of naturally occurring constituents, microorganisms, pesticide residues, antibiotics, and fragments of microbial constituents [6]. The majority of food constituents, such as sugar, acid, protein, and alcohols can be determined using enzymes, except food additives such as antioxidants and preservatives. Enzymes are used to perform routine analytical procedures that were laborious in the past; and they are used to determine the primary, tertiary, and quaternary structures of polymeric molecules, such as proteins, nucleic acids, carbohydrates, and triglycerides. In this context, the role of enzymes as analytical instruments for food analysis and safety is discussed in this chapter.

10.2 ENZYME INDICATORS FOR MILK PASTEURIZATION Heat treatment is a critical step for shelf life extension and the safety of milk and milk products. For rapid validation of the milk pasteurization process, an alkaline phosphatase (ALP)

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test has been adopted by many counties as a standard method. Efficiency of pasteurization (72°C, 15 s) is determined by the absence of ALP activity in heat treated milk, which is an accepted indicator to determine the efficiency of the pasteurization of milk. This test is based on the thermal inactivation characteristics of ALP enzymes naturally present in milk. In other words, the resistance of ALP is slightly more than that of the target bacterial pathogens (Mycobacterium tuberculosis and Coxiella burnetii, the heat resistant bacterial in milk); thus, if reduction in ALP activities was observed, it indicated that the target bacterial pathogenic population was similarly reduced, and the required thermal processing for pasteurization was met [7]. Estimation of ALP activity requires incubation of milk with either fluorogenic (benzothiazole orthophosphoric monoester), chromogenic (p-nitro-phenyl phosphate), or disodium phenyl phosphate [8]. Instead of milk, this test has also been used as a marker for ascertaining proper heat processing of milk-based products such as milk powder, cheese, cream, ice cream, and so forth. The other enzymes indigenous to milk, such as lactoperoxidase and γ-glutamyl transpeptidase, have been identified as potential indicators of the heat processing of milk. Lactoperoxidase and γ-glutamyl transpeptidase are inactivated at 80°C in liquid milk, and at 90°C in an ice cream mix and cream [9,10]. Alkaline phosphate is a protein that, remarkably, occurs in nature and in many human body tissues such as the liver, kidney, blood cells, bones, and so forth. It is available in milk and body fluids from many organisms at different levels. ALP is a glycoprotein bound to a membrane with sialic acid as a sugar moiety. It is a phospho-monoesterase enzyme that yields phosphate and alcohol by catalyzing the hydrolysis of monoesters of phosphoric acid in an alkaline environment. Due to its widespread occurrence and importance in biological systems, ALP activity assessments are recognized as the most important, and common, enzymatic assay [7]. ALP activity can be detected by four methods: colorimetric, fluorometric, chemiluminescent, and immunochemical. A general procedure for the assay of milk for ALP activity is shown in Fig. 10.1. For every assay, it is necessary to run positive and negative controls, and follow the procedures as per availability of materials. Based on the activity of γ-glutamyl transpeptidase and lactoperoxidase, rapid indicative methods were developed for the assessment of the effectiveness of the heat processing of milk at 80°C for 15 s. This heat processing completely inactivates both milk enzymes. These simple methods differentiate between milk samples heated at 80°C for 15 s and those heated at 75°C or lower. The developed methods can be applied in a quality control laboratory [8]. A colorimetric assay as a means of testing ALP activity is based on the liberation of phenol from disodium phenyl phosphate, p-nitrophenyl phosphate, or phenolphthalein monophosphate as a substrate. The use of phenyl phosphate as a substrate to estimate ALP activity employs the detection of phenol released by a secondary colorimetric reaction, and ALP activity is expressed as an amount of phenol equivalent produced per unit of sample (the Kleyn method). The standard reference method for ALP measurement is the fluorimetric method. In this assay, a fluorescent compound, fluoro yellow, is produced by hydrolysis of the fluorophos substrate, and then analyzed using a fluorometer. The instrumental measurement of fluorescence generated over time is correlated to enzymatic activity, or concentration of ALP [11]. The ALP activity is expressed as mU/L, a unit of activity of ALP that expresses the amount of enzyme that catalyzes the transformation of 1 μmol of substrate per minute.

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FIG. 10.1 A general procedure for assay of milk for ALP activity.

Lactoperoxidase is another milk enzyme naturally present in milk that is more heat-stable than ALP. It is inactivated at 75–80°C; therefore, if milk is exposed to a higher temperature (>75°C) during pasteurization, the enzyme is inactivated, and a negative peroxidase (POD) test will result. The method for determination of the lactoperoxidase activity depends on the ability of POD to oxidize hydrogen peroxide: oxygen is developed that oxidizes the colorless 1,4-phenylenediamine into the purple indophenols, with the color intensity proportional to the enzyme concentration. If the milk is properly pasteurized, a blue color will occur within 30 s after mixing; otherwise, no color will occur, which means lactoperoxidase has been inactivated [12].

10.3 ENZYME INDICATORS FOR VEGETABLE BLANCHING Blanching is an important and essential step usually performed prior to many preservation processes such as canning, drying, frying, and freezing. The main objectives of blanching are to inactivate enzymes such as polyphenol oxidase (PPO) and POD, and to prevent distorted

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flavor, undesirable color changes, and other possible deterioration reactions. Other objectives include reducing the microbial population for shelf life extension, increasing the rate of heat and mass transfer by eliminating air, softening tissues for better texture, and preventing oxidation [4]. In blanching, the requirement is to provide adequate heat treatment to stabilize the product against quality deterioration, and to minimize quality loss. These benefits of enzyme inactivation by blanching help it to act as an indicator of adequate heat treatment. PPO, POD, and catalase are known to be resistant to heat, and are widely used as the indicators of adequacy of blanching. The disappearance of certain enzyme activities during blanching correlates with extended shelf life. Process optimization is based on determining the rate of enzyme destruction, so that the blanch time is just long enough to destroy the indicator enzyme. Many food processors employ a heat treatment high enough to inactivate POD, one of the highly resistant enzymes present. Some researchers recommend that targeting POD inactivation results in more severe heat treatment than is required for many vegetables, and the enzyme responsible for quality deterioration has a lower stability than POD [13,14]. Blanching indicators in frozen food can be identified as [15]: (i) blanching food using various time-temperature combinations, (ii) freezing preblanched food and storing it for a known time, (iii) thawing and evaluating quality by a sensory panel, (iv) testing the food for a range of endogenous enzyme activities, and (v) identifying the correlation between enzyme activity and sensory scores.

10.3.1 PPO and POD as Indicators of Blanching The efficiency of blanching is generally determined by the inactivation degree of PPO and POD because they are easy to measure. PPO catalyzed an enzymatic browning, which is generally considered deleterious to food quality from a nutritional and sensory point of view. Because of this, PPO has been considered and studied as a target during the blanching process. Endogenous hydrogen peroxide in vegetables can react with POD to produce free radicals that react with a large range of food constituents, such as fatty acids, ascorbic acid, and carotenoids. This brings about undesirable changes in food products such as color, flavor, and nutrient degradation [16]. POD requires a long blanching treatment for complete inactivation, as it is the most heat resistant enzyme. The inactivation of POD could cause a great loss of nutrients, and increase the cost of energy [17]. Nurhuda et al. [18] conducted steam and water blanching of rambutan peel extract to determine the residual POD and PPO activities. They reported that both steam and water blanching significantly reduced POP and POD activities, and suggested that with the increase in the blanching period, enzyme activities did not reduce further. The best blanching conditions for inactivation kinetics of PPO, POD, and inulinase of a garlic clove cut in slices were steaming for 4 min, which resulted in no change in texture; and the enzymatic activities were reduced by 92.15%, 93.53%, and 81.96% for PPO, POD, and inulinase, respectively [19]. The effect of ultrasonic waves (thermosonication) and heat on the inactivation kinetics of POD in seedless guava revealed that thermosonication inactivates POD in seedless guava at lower blanching conditions, and the rate of inactivation was increased by 1.5–3 times in the temperature range of 80–95°C, with 50%–75% ultrasonic wave amplitudes [20]. The effect of microwave blanching (MWB), hot water blanching (HWB), high-humidity hot air impingement blanching (HHAIB), and infrared blanching (IRB) on the enzyme

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inactivation of the PPO and POD of red bell peppers was investigated. The results depicted that all four blanching treatments had, remarkably, inactivated the enzymes of red bell pepper. The remaining activity of POD and PPO were 16.43% and 9.8%, respectively, at high microwave treatment (900 W) blanched for 100 s [4].

10.3.2 Heat Stability of Indicator Enzymes Heat inactivation of enzymes responsible for quality deterioration in fruits and vegetables demands proper determination of temperature stabilities. Thermal stabilities of enzymes varied from different sources. Therefore, it is important to discover the heat stability of indicator enzymes for different vegetables before conducting a suitable blanching process. The variation in heat stability of indicator enzymes in various foods requires the processor to observe the time needed for each food, with the blanching apparatus and conditions used. Thermal inactivation of enzymes may be considered as a first-order monophasic model, or second-order biphasic model. The first-order model is established on the assumption that to inactivate the enzyme, cleavage of a single bond is sufficient. A biphasic model is based on the analysis of thermal inactivation kinetics of an enzyme mode formed by two groups that differ in their heat stability [21]. POD is more resistant to heat in low-acid foods than in more acidic foods. The thermal inactivation kinetics of POD in a butternut squash were determined by immersing it in water at 60–90°C for 0–60 min [22]. A time-temperature binomial was observed for inactivation of POD in mate leaves by using a conveyor belt oven. POD exhibited higher heat resistance than POP. The consecutive step models and biphasic models revealed that the best inactivation of POD was obtained by treatment at 255°C for 20–24 s [23]. Heat inactivation curves for POD in coriander leaves were observed in the temperature range of 70–100°C with steam. The best model to describe POD inactivation kinetics in coriander leaves was a two-fraction, first order model (R2 > 0.97) [21]. The kinetics of inactivation of POD in mangosteen peel were studied for a temperature range of 60–100°C. The inactivation kinetics pursued a monophasic first order model, having k values between 1.93  10 2 and 8.14  10 2 per min. The reducing trend of k value with increasing temperature indicated a rapid inactivation of POD from mangosteen carp at high temperatures [24]. Bai et al. [25] used PPO as the indicator enzyme to study an apple’s quality under highhumidity air impingement blanching. PPO was entirely inactivated within 7 min at 90–120°C, and this inactivation followed a zero-order kinetics model at 90°C and 100°C, and followed a first-order fraction model at 110°C and 120°C. Therefore, apple quality was maintained by inactivating PPO at faster rates, and the high humidity air impingement blanching process was observed to be an effective pretreatment.

10.4 BIOSENSORS AS AN ANALYTICAL TOOL IN FOOD QUALITY AND SAFETY Monitoring safety and quality of food is an essential part of the food industry. Biosensors are diagnostic tools that are becoming increasingly favorable in food analysis. These provide

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excellent substitutes for methods that are more energy- and time-consuming [26]. A biosensor is an instrument composed of a biospecific recognition system (cells, enzymes, antibodies, proteins, etc.). This instrument is used to measure the concentration of target analytes. For this purpose, biosensors work in combination with a physicochemical transducer that transforms the biotic reaction into a quantifiable signal. The extent of this quantifiable signal is directly proportional to the concentration of the target analyte. A transducer transforms this biotic reaction into an electric signal. Therefore, the properties of transducers matter the most in the proper functioning of biosensors. The most widely used biosensors are based on the electrochemical properties of transducers and analytes. The most common types of biosensors rely on tracking analytes with enzymes. The high applicability of enzymatic biosensors is because of their rapid response, sensitivity, availability of number of commercial enzymes, and methodologies in the construction of these biological sensors. Enzymatic biosensors rely on different methods of immobilization. The most commonly and specifically used enzymes for this purpose are aminoxidases, PPO, oxidoreductases, and so forth [27,28]. The list of major enzymes used in enzymatic biosensors is provided in Table 10.1.

10.4.1 Principle of a Biosensor The fundamental principle of a biosensor depends on three strands: appliances that are used for constructing biosensors, molecular remembrance segments, and transducers. The transducer, the main component in biosensor design, is classified according to the physical change accompanying the reaction [29]. Enzymes are immobilized by conventional methods. TABLE 10.1 Enzymes Used in Biosensors to Detect Food Components Food Components

Enzyme

Glucose

Glucose oxidase, glucose dehydrogenase

Fructose

Fructose-5-dehydrogenase

Sucrose

Glucose oxidase, mutarotase, invertase

Lactose

Galactose oxidase, peroxidase

Glutamine

Glutaminase

Malate

Malate dehydrogenase

Galactose

Galactose oxidase

Cholesterol

Cholesterol oxidase

Alcohol

Alcohol oxidase

Oxalate

Oxalate oxidase

Tyrosine

Tyrosinase

Aspartame

Peptidase, glutamate oxidase

Phenols

Tyrosinase

Essential fatty acids

Lipoxygenase

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A bound analyte generates the signal that is measured by the biosensor. Based on output, biosensors can be classified as: (i) calorimetric biosensors: heat output/absorbed by the reaction, (ii) electrochemical biosensors: electrical/electronic output, (iii) amperometric biosensors: redox reactions, (iv) optical biosensors (absorbance, fluorescence, chemiluminescence): light output/absorbance, (v) Piezo-electric biosensors: acoustics/sound vibrations, and (vi) immuno biosensors: immunological specificity.

10.4.2 Enzyme Immobilization Enzyme immobilization is a very important element in the proper functioning of a biosensor. The choice of specific enzyme immobilization method relies on proper deposition of the enzyme, chemical inertness of transducer’s surface, and so forth. Enzyme immobilization can be achieved by several methods, such as adsorption, entrapment, covalent binding, and crosslinking [30]. The major enzyme immobilization methods are summarized as follows. 10.4.2.1 Physical Adsorption Onto a Solid Surface Adsorption represents a simple, quick, and easy way of depositing an enzyme on a transducer surface. Two different types of adsorption include: physical adsorption (van der Waals bonds), and chemical adsorption (covalent bonds). Physical adsorption is one of the oldest immobilization methods that relies on van der Waals forces, hydrogen bonds, and ionic interactions between the enzyme and transducer. The simplicity, and the types of beads utilized in this method make this technique distinctive among others [31]. 10.4.2.2 Cross-Linking With Bifunctional or Multifunctional Reagents In this method, electrodes are immersed in an enzyme infusion for a fixed time. After a specific period of time, enzymes are deposited on electrodes. Then, this treatment is followed by exposing the enzyme-deposited electrodes onto glutaraldehyde for cross-linkage [32]. 10.4.2.3 Entrapment in a Matrix Physical entrapment of enzymes into conducting polymers is a popular technique. Entrapment means physical enclosure of a biomolecule in a small space. Inert membranes are used to provide close contact between biomaterials and the transducer. Types of membranes used include cellulose acetate, polycarbonate, collagen, and Teflon. The entrapment method for enzyme immobilization involves bulk entrapment in a polymer matrix that remains on the electrode surface from the solution containing the dissolved monomer and enzyme. The embedment of enzymes within a conducting polymer film prevents the enzyme from being leached out, and maintains the accessibility of the catalytic sites because of porosity of the film to analytes. This immobilization method is widely used for amperometric biosensors, and to a lesser extent, for other electrochemical transducers, such as conductimetry and potentiometry [33]. 10.4.2.4 Covalent Binding to a Surface Covalent bonding is another method of enzyme immobilization in biosensors, in which some functional groups are covalently bonded to the support matrix. In this method,

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nucleophilic groups (amino, carboxyl, sulfhydryl, hydroxyl, phenolic, and thiols) present in amino acids of the enzymes are used to form covalent linkage. The reaction is carried out under specific conditions, that is, at low temperature, neutral pH, and low ionic strength in order to avoid the loss of functional enzymes. This technique is successful in significantly improving the lifetime of biosensors [34]. 10.4.2.5 Sol-Gel Entrapment The latest application of sol-gel-derived materials is in the area of immobilization of enzymes. Sol-gel formation is an easy method that generates a stable material where enzyme activity is preserved, and sensitivity of biosensors is enhanced. The sol-gel networks prepared by a generalized synthesis involve acid or base catalysts, and an aqueous or no aqueous solvent. Three steps are involved in sol-gel formation; namely, hydrolysis of the silicate, condensation, and polycondensation. Sol-gel methods used for generating nanomaterials in biosensor applications have been developed recently [35]. 10.4.2.6 Electrochemical Polymerization Polymeric films are the most commonly reported matrix for immobilization of enzymes. Different polymeric films immobilize enzymes by ion pair interactions and hydrogen bonds, and stabilize them by shielding active sites from a polar aqueous environment, and diminishing the water activity around the protein. Polymeric films are designed as conductive, nonconductive, and composite, and are fabricated by solvent casting, electro polymerization, and spin coating [36]. 10.4.2.7 Nanomaterial Immobilization Nanomaterials provide opportunities for the development of the novel design of biosensors. Carbon nanotubes, nanowires, magnetic nanoparticles, nanorods, and quantum dots have a high capacity for charge transfer, and they increase the sensitivity and detection limits. These nanomaterials combine with the graphitic structure chemistry, and confer electronic properties to carbon nanotubes that make them an ideal material for use in chemical and biochemical sensing. The use of metal nanoparticles and grapheme is a common practice for increasing surface area and conductivity of the electrochemical biosensor [37].

10.4.3 Applications of Enzyme Biosensors in Food Quality and Food Safety Monitoring safety and quality of foods is essential for the food industry. The use of biosensors helps to determine food safety in a very safe and efficient manner. Biosensors act as a useful alternative as compared with other methods. This can be easily visualized from their operation in a very short duration. The biosensors have a wide range of applications in food quality and safety, including: (i) quantification of different food components to evaluate rancidity, maturity, and shelf life; (ii) detection of substances that are used as indicators of food freshness; (iii) quantification of compounds found in low or high concentrations (e.g., sugars, amino acids, acids, alcohols); (iv) implementation of hazard analysis and critical control points (HACCP) plans by verifying processes and correcting critical points in due time; (v) detection of total microbes in different

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foods; (vi) food quantification in soft drinks; (vii) food decay detection; (viii) testing of foodstuffs for maximum pesticide residue verification through routine analysis of analyte concentrations, such as, glucose, sucrose, alcohol, and so forth; (ix) testing the quality of waste water; (x) assessment and analysis of produce such as wine, beer, and yogurt; (xi) to detect small molecules, such as water-soluble vitamins and chemical contaminants in food stuffs; (xii) detection of any pathogenic organisms present in meat, poultry, eggs, and fish; (xiii) determination of drug residues in food; (xiv) detection of contaminants such as Salmonella, Listeria, and Staphylococcus in meat and dairy products; (xv) monitoring and control of fermentation processes; (xvi) detection of olfactory qualities and flavor; (xvii) analysis of fats, proteins, and carbohydrates in food; (xviii) detection of rancidity in food stuffs; (xix) monitoring of raw materials conversion; (xx) detection of allergens in food stuffs; and (xxi) verification of product contents. 10.4.3.1 Applications in Food Quality Food quality is characterized by a combination of organoleptic and nutritional attributes [38]. Freshness of food is affected by several factors, such as improper temperature, temperature fluctuations, improper time of preservation, wrong selection of food package, and so forth. In the case of alcoholic beverages, quality and freshness is indicated by methanol and ethanol. The various applications of sensors applied in the food industry for food quality estimation are tabulated in Table 10.2. The sensory evaluation of “Idiazabel” cheese has been proposed by Perez et al. [38]. Their study has been applicable to any type of food and beverage. Expert sensory panelists can be easily replaced by biosensors in an effective manner. 10.4.3.2 Applications in Food Safety Different emerging areas of food quality management systems include food safety, and quality control and assurance. Continuous improvement in the whole scenario is an important part of food quality management systems, which should not to be ignored in any circumstances. High sensitivity of biosensors provides rapid results for detecting microorganisms, foodborne pathogens, impurities, adulterants, and so forth. HACCP (Hazard Analysis Critical Control Point) is an important food safety management tool that is used for tracing processes and risks involved with any system in a controlled manner [39]. High sensitivity of biosensors, in combination with HACCP, is a promising approach for overseeing bacteria in-line. Detection of bacterial counts is possible by developing low-cost biosensors [40]. Enzymatic biosensors are the most popular and convenient among various kinds of biosensors. They are the most effective, convenient, rapid, and sensitive devices when they utilize nanomaterials for further advancement in technology [5]. Electrochemical biosensors are based on a synergistic approach that utilizes enzymes and nanomaterials for diagnosis of several food safety issues. The applications of biosensors in food safety are summarized in Table 10.3. 10.4.3.3 Applications in Smart Packaging Smart packaging is a novel technology in the food industry. This technology involves builtin-sensors and indicators to supervise food quality and freshness. This technology deals with better supervising and communication about the quality of food in an entirely different

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TABLE 10.2

Enzymatic Sensors Applied in Food Industry

Analyte

Applications

Enzyme

Transducer

Fruits and vegetable processing Methanol

Beer, wine

Alcohol oxidase/horse radish peroxidase

Amperometric

Caffeine

Instant tea and coffee

3,5-Cyclic phosphodiesterase

Amperometric

Oxalate

Spinach

Oxalate oxidase/horse radish peroxidase

Amperometric

Glutamate

Tomato foods

Glutamate oxidase

Amperometric

Glucose

Juice

D-Glucose

Amperometric

Glucosinolates

Vegetables

Glucose oxidase

Amperometric

Ethanol

Wine

Alcohol dehydrogenase

Optical

D-Malate

Juice

D-Malate

Optical

L-Glutamate

Tomato

L-Glutamate

Acetic acid

Wine

Acetate kinase

Amperometric

Ascorbic acid

Juice

Ascorbate oxidase

Calorimetric

L-Amino

Juice

L-Amino

Amperometric

Fructose

Honey, apple juice

–

Amperometric

Polyphenols

Red wine

Laccase

Amperometric

Monophenols, dihydroxyphenols

Mushrooms

Tyrosinase

Amperometric

Cholesterol

Butter

Cholesterol oxidase

Spectrofluorometric

Urea

Milk

Urease

Potentiometric

Listeria monocytogenes

Milk

–

Electrochemical

Cholesterol

Fish

Cholesterol oxidase and esterase

Amperometric

Xanthine

Fish

Xanthine oxidase

Amperometric

Xanthine

Meat and fish freshness

Xanthine oxidase, superoxide dismutase, and peroxidase

Fluorescent

Hypoxanthine

Fish freshness

Xanthine oxidase

Amperometric

Histamine

Fish and meat

Monoamino oxidase

Amperometric

acid

dehydrogenase

dehydrogenase dehydrogenase

acid oxidase

Amperometric

Milk and milk processing

Meat processing

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TABLE 10.3 Enzymatic Sensors Applied in Food Safety Analyte

Application

Enzyme

Transducer

Natural toxin

Potato

Butyl cholinesterase

Potentiometric

Natural toxin

Potato

Acetyl cholinesterase

Potentiometric

Azide

Fruit juice

Catalase

Amperometric

Pesticide

Wheat

Choline oxidase

Amperometric

Pesticide

Infant food

Acetyl cholinesterase

Amperometric

Mercuric ions

Drinking water

Urease

Optical

Carbaryl and dichlorvos

Drinking water

Choline esterase

Optical

Chlorpyrifos

Food sample

Acetyl cholinesterase

Optical

Nitrate

Food sample

Nitrate reductase

Potentiometric

Phosphate

Milk

Pyruvate oxidase

Amperometric

Hg, Ag, Pb, and Cd

Water and food

Invertase and glucose oxidase

Ultramicroelectrode

Cu and Hg

Food sample

Glucose oxidase

Amperometric

Paraoxon

Cucumber

Acetyl cholinesterase

Carbon nanotubes

Methyl parathion

Garlic

Organophosphorus hydrolase

Graphene

Malathion, chlorpyrifos, monocrotophos, endosulfan

Milk and water

Acetyl cholinesterase

Multiwalled carbon nanotubes

Escherichia coli

Surface water

–

Electrochemical

Salmonella typhimurium

Chicken carcass wash

–

Electrochemical

Listeria monocytogenes

Milk

–

Electrochemical

Campylobacter jejuni

Culture and chicken carcass wash

–

Electrochemical

manner. The product’s history in the food supply chain can be traced with the advent of smart packaging. Different types of sensors and indicators play a wide role in the success of smart packaging. These include: time temperature indicators (TTIs), freshness indicators, integrity indicators, and radio frequency identification (RFID) [41]. A freshness indicator is usually in the form of labels that are applied on containers. These indicators work on the principle of change of pH or gas composition, which are detected by indicators and transformed into a response; usually a color response that can be easily measured [42]. The label sensor is based on a methyl red cellulose membrane that increases the pH due to volatile amine decomposition. This technology is successfully implemented in the monitoring of the fresh meat of boiler chickens [43]. TTIs are based on judging the temperature’s history in the environment in which the product is placed. This results in a color change that truly relies on various polymeric reactions,

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including mechanical, photochemical, and so forth [44]. The first generation TTIs initially used lipase enzymes to stipulate color changes that were pH dependent due to lipid hydrolysis [45]. TTIs have divergent applications in food industries that deal with fruits and vegetables, meat products, dairy products, fisheries, and so forth [41]. The electrochemical biosensor is the foremost instrument that monitors food quality and freshness in a very smart manner. It is an important sensor in smart packaging technology. Several advantages are associated with these biosensors, including small size, no additional instrumentation requirement, easy handling, and so forth. These biosensors are substratespecific, and no pretreatment is required for the functioning of these sensors [46]. RFID tags are an advanced form of data facts that can reveal and distinguish a product with a unique tag that releases radio waves. Each RFID tag applied to a packaging container transmits the information to a reader, which is passed to a computer [47]. These devices can be coupled to an article, container, box, or pallet for identification and tracking. The RFID system is composed of two main elements: the tag and reader. Readers are the stable, handheld computers that are placed in deliberate areas. The wide application areas of RFID tags include estimating the freshness of foods such as cod fish and meat, monitoring temperature and humidity, and so forth [48].

10.5 ELISA AS AN ANALYTICAL TOOL IN FOOD QUALITY AND SAFETY ELISA, a plate-based assay technique, is a very distinguishable and unique method for identifying and tracing the presence of antigens or antibodies in a sample. This technique is performed by utilizing 96-well or 384-well polystyrene plates that progressively tie antibodies and proteins. Detection is the most important and sensitive part of ELISA, which is proceeded by judging the conjugated enzyme activity through incubation with a substrate to generate a colored product.

10.5.1 Principle of ELISA The basic principle of the ELISA (a plate-based assay) technique is to recognize the coupling of an antigen with antibody in a very unique manner. Enzymes play a lead role in this process, as it depicts a color change by converting a colorless substrate to colored products; thereby indicating the coupling of an antigen with an antibody (Fig. 10.2). The basic principles involved in ELISA are as follows. 10.5.1.1 Antigen-Antibody Reaction Whether an antigen or an antibody is present in a sample, it is detected by means of ELISA. An antibody (Ab) is coupled with an antigen (Ag) in a very systematic manner to generate an Ag-Ab complex. 10.5.1.2 Enzymatic Chemical Reaction Quantification of an antigen or antibody is directly coupled with the formation of an Ag-Ab complex. Different enzymes are used for labeling antibodies. These enzymes include

286 FIG. 10.2

10. ENZYMES AS TOOLS FOR ASSESSMENT OF FOOD QUALITY AND SAFETY

Principle of ELISA.

horseradish POD, ALP, β-galactosidase, lactoperoxidase, and so forth. Enzymes play an important role in this process as it results in the generation of colored products. 10.5.1.3 Signal Detection and Quantification Detection of signals occurs when both the enzyme and substrate are added onto the process. This process is followed by a color change, which helps in quantification of the results. The overall process is more pronounced by the generation of color and its intensity. The different steps or principles of ELISA are presented in Fig. 10.2.

10.5.2 Procedure of ELISA The general procedure of ELISA includes various steps such as coating, blocking, detection, and reading of results. Each step plays a significant role in the functioning of ELISA. Coating is the initial step, in which adsorption occurs on a polystyrene plate, followed by the blocking step. In the blocking step, a blocking agent is used for coating the unbound sites. This step is followed by the incubation of plates with antibodies. The last step is detection, in which a color change indicates the success of ELISA. The various steps of ELISA are presented in Table 10.4.

10.5 ELISA AS AN ANALYTICAL TOOL IN FOOD QUALITY AND SAFETY

TABLE 10.4

287

Procedure of ELISA

S. No.

Step

Procedure

1

Coating

• Initial step • In this step, adsorption of target antigen or antibody occurs onto a 96-well polystyrene plate

2

Blocking

• • • •

3

Detection and reading of results

• Most crucial step • A calorimetric signal is generated by adding substrate • Detection of antibodies that are further labeled by application of enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP)

In this step, coating plays an important role Blocking agent is coated onto all unbound sites Incubation is the next essential step Incubation of 96-well polystyrene plate is done with an antibody (enzyme conjugated) • The preceding step proceeds after repeated washing; resulting in removal of all unbound antibodies

10.5.3 ELISA Kits High-quality ELISA kits utilize biologically relevant materials for providing consistent results with any sample type. Dissimilar ELISA kits are accessible depending upon their use and application. Nonidentical types of ELISA kits are obtainable for use in food safety and quality. These include food allergy self-test kits, Tepnel peanut kits, biokits, enrofloxacin ELISA test kits, and so forth. Various types of ELISA kits have wide applications for detecting antigens, antibodies in serum, and in tracking outbreaks of certain diseases. The various features of ELISA kits include accuracy, versatility, value, technical support, sensitivity, and specificity.

10.5.4 Applications of ELISA in Food Quality and Food Safety An immunoassay technique such as ELISA has a strong correlation with the food sector. ELISA has been widely used for identifying microorganisms, naturally occurring and microbial constituents, pesticide residues, and antibiotics. ELISA plays a major role in areas such as food safety, analysis, processing, and production [6]. The applications of ELISA in the food industry are summarized in Table 10.5.

10.5.5 Applications of ELISA in Food Safety and Quality ELISA is an excellent qualitative tool for detecting contamination and adulteration in different food stuffs. It has wide applications in food safety and quality. For this purpose, various food safety ELISA kits are available. The list of various ELISA kits used for food safety and quality, along with their specific uses, are presented in Table 10.6.

TABLE 10.5 Applications of ELISA in the Food Industry Type of ELISA

Applications

Food Product

Reference

Indirect competitive and Sandwich ELISA

Illicit incorporation of low quality bovine milk in cheese preparation

Milk and milk products

Zachar et al. [49]

Direct and Indirect ELISA

Fraudulent switching of slashed fish species in eminent quality fish

Fishery products

Gordoa et al. [50]

Sandwich ELISA, indirect ELISA

Detection of adulterants in ground and comminuted products

Meat and meat based products

Ayaz et al. [51]

Sandwich ELISA, indirect ELISA

Screening of a distinguished GMO in raw materials

Genetically modified (GM) foods

Khetarpal and Kumar [52]

Direct and indirect ELISA

Determination of animal origin based constituents

Feed stuffs

Nesic et al. [53]

Competitive ELISA

Detecting allergens and aflatoxins in numerous food products

Wheat, peanuts, milk, shellfish, eggs, soybeans

Leszczynska et al. [54]

Direct and indirect ELISA

Quantification of amygdalin (capable of generating hydrogen cyanide)

Apple seeds, processed apple juices, and fresh apples

Bolarinwa Islamiyat et al. [55]

Sandwich ELISA

Distinction of residues of lupine in different food items

Corn muffins, beef frankfurter, and apple cinnamon muffin

Kaw et al. [56]

Sandwich ELISA

Detection of mustard in food stuffs

Baked bean products

Lee et al. [57]

Sandwich ELISA

Identification of cashew nut in dairy and bakery food stuffs

Ice cream, cookies, and milk chocolate

Gaskin and Taylor [58]

Sandwich ELISA

Identification of walnut residues in different food stuffs

Ice cream, milk chocolate, muffins, and cookies

Niemann et al. [59]

Sandwich ELISA

Recognition of buckwheat residues in cereal based foods

Noodles and muffins

Panda et al. [60]

Direct and indirect ELISA

Detection of food authenticity

Processed food products and feed stuffs

Scarano and Rao [61]

TABLE 10.6 ELISA Kits for Food Safety and Food Quality Kit

Company

Country

Uses

Allerquant 14 food additive

Biomerica

California, United States

Food allergen testing

Antigliadin IgA ELISA kit

Biomerica

California, United States

Celiac disease

Clonidine, phenyl ethanolamine, cyproheptadine, oxytetracycline, nitroimidazole ELISA kits

Life Technologies

New Delhi, India

Antibiotic residue testing kits for animal feed

Transglutaminase IgA ELISA

Biomerica

California, United States

Food intolerance

Allerquant Med90G ELISA

Biomerica

California, United States

Food allergen testing

289

10.6 CONCLUSIONS AND PERSPECTIVES

TABLE 10.6

ELISA Kits for Food Safety and Food Quality—cont’d

Kit

Company

Country

Uses

Listeria Tek and Tecra

Bioline

London, United Kingdom

Detecting listeria in naturally contaminated foods

Oxytetracycline, ractopamine, salbutamol, terbutaline, tetracycline, trimethoprim, tylosin ELISA kits

Life Technologies

New Delhi, India

Antibiotic residue testing kits for meat and meat products

Salmonella ELISA kit

Bioline

London, United Kingdom

Meat, seafood, fish, egg, water, and milk

Histamine detection kit

LDN (Labourdignostika Nord)

Nordhorn, Germany

Determination of histamine in fish meal, milk, cheese, sausage, wine, and fresh fish

ELISA kit

ID Biotech

Issoire, France

Detection of bovine gelatin in porcine gelatin

Benzopyrene, benzyl penicillin, ciprofloxacin, dexamethasone ELISA kits

Life Technologies

New Delhi, India

Antibiotic residue testing kits for fish and fish products

ARILAIT ELISA kit

ID Biotech

Issoire, France

Measurement of alkaline phosphatase from bovine milk

Benzyl penicillin, chloramphenicol, ciprofloxacin, fluoroquinolone, neomycin ELISA kits

Life Technologies

New Delhi, India

Antibiotic residue testing kits for milk and dairy products

Calbiotech Brucelle IgG ELISA kit

CALBIOTECH

Dubai, United Arab Emirates

Detection of immunoglobulin antibody to Brucella in human serum or plasma

Dimetridazole, nitroimidazole, norfloxacin ELISA kits

Life Technologies

New Delhi, India

Antibiotic residue testing kits for egg and egg products

10.6 CONCLUSIONS AND PERSPECTIVES Food quality and safety is a prime criterion for everyone; whether it is a consumer or a manufacturer. Numerous enzyme-based analytical tools exist to maintain food quality and safety. Biosensors emerge as one of the best diagnostic tools among other technologies in the area of food quality and safety. Enzymatic biosensors are generally used in the food industry to determine freshness of products, to control the fermentation process, to control acidity, and for assessing thermal profiles. Various properties of biosensors, particularly optical, mechanical, and electrochemical, can be modified by the application of nanomaterials. This will create a new era in the field of biosensors. In milk processing, heat treatments represent the common practice for inhibiting microbial growth and detecting the adequacy of heat treatment.

290

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The ALP method has been shown to be a remarkably valuable tool for the routine assessment of milk pasteurization validation. Blanching is a pretreatment that plays a distinguishable role in the processing of fruits and vegetables. The indicators used to judge the adequacy of blanching are POD and PPO enzymes. ELISA is a powerful tool for sensing and judging the presence of specific analytes within a sample. Different type of ELISA such as direct, indirect, sandwich, and competitive vary in some aspects. But in general, ELISA methods coupled antibodies to the antigens; and then used an enzyme-linked conjugate to generate colored products that can be detected. ELISA kits have wide applications in the food sector for detecting naturally occurring microbial constituents, antibiotics, pesticide residues, and so forth. Enzymes thereby provide an excellent screening tool to detect food quality, adulteration, and contamination; indicating their important role in food safety and quality. But the gap between marketplace and research is still wide, and commercialization of enzyme-based analytical technologies needs to be focused with the advent of technology.

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