eChapters

1. Chlorophylls

Chlorophyll can be regarded as a true representation of health of green plants. This presumption is based on the fact that chlorophyll is most commonly and immensely influenced by biotic and abiotic stress conditions in addition to events like pathogenic invasion, pollution, mineral deficiencies as well as mineral toxicity. This is the reason that estimation of chlorophyll is considered to be one of the most important parameters in plant physiological studies.

Eukaryotic plant cells possess many types of ‘reserves’, one of them is plastid. A plastid, that contains green colour is known as chloroplast and the green colouration of a chloroplast is mainly due to the presence of a pigment to which we call as chlorophyll. Normally, chlorophyll constitutes about 4% of chloroplast dry weight.

Chlorophyll ‘a’ and ‘b’ are the most abundantly found in all autotrophic higher plants. However, there are other chlorophylls also such as ‘c’, ‘d’, ‘e’, bacteriochlorophyll and bacterioviridin which have restricted presence in blue-green, brown and red algae.

A chlorophyll molecule contains ‘Tetrapyrrole’ skeleton formed into a ring with an atom of magnesium (Mg⁺⁺) in the centre. A so-called ‘Pyrrole’ molecule contains four carbon and one nitrogen atoms arranged in a ring. Four such pyrrols arranged in a ring form the ‘Head’ of chlorophyll molecule. Attached to this is ‘porphyrin ring’, at one point, is a ‘phytol’ tail- a long chain of linked carbon.

The weight of living plant material contained above and below a unit of ground surface area at a given point of time, is known as plant “Biomass”. Biomass is of utter importance because it provides energy source (Roberts et al. 1987).

Biomass, which has dimensions of mass per unit, is expressed as g m^-2, kg ha^-1 or t ha^-1.

Measurement of Biomass

The area (in the crop field) under study is divided into a number of equal squares or rectangular plots measuring 1.0 m² or 0.5 m². Each square is designated with a particular number. Sample can be harvested from a small area or quadrate at the centre of each square; the remaining peripheral area serving as buffer zone (Wiegert, 1962).

Depending upon the growth pattern of plant species two different designs have been adopted for the sampling:

Growth represents overall performance of a plant under a particular environment. Generally, we restrict to the above ground plant parts for growth studies, however, a better understanding of plant growth and development would be achieved if total dry matter (including above ground shoot as well as underground root) were taken into consideration.

The basic principles behind growth analysis constitutes (i) data on plant height and (ii) leaf area. The quantitative analysis of growth is based upon several terms, which are described below (Hunt, 1978).

1. Growth

Growth is an irreversible increase in size which may or may not be accompanied by an increase in dry weight. It is the sum total of all the metabolic activities and normally results from the excess of anabolism over catabolism. In general, it refers to quantitative changes in the life cycle of plants. However, there are a few examples where growth is not accompanied by an increase in dry weight such as (i) germinating seeds and, (ii) etiolated plants.

1. Relative Water Content

Relative water content (RWC) is an important parameter to measure the water status of plant tissue. High RWC, under moisture stress, denotes ability of plants to tolerate drought. (Ritchi et al. 1990). RWC and water potential have often been reported as indicators of drought tolerance (Schonfeld et al. 1988).

Leaf water status may be estimated by measuring the RWC following Barrs and Weatherlay’s (1962) method as given below:

The most abundantly and commonly available compounds in the plant kingdom are carbohydrates. They comprise saccharides viz.

Monosaccharides - glucose, fructose etc.

Disaccharides - sucrose, lactose, maltose etc.; and

Polysaccharides - starch, cellulose etc.

Cellulose is the major component of plant cell wall.

The active groups in carbohydrates are aldehyde (-CHO) and ketone (=CO). In addition, they contain hydroxyl groups as well. The chemical properties of saccharides vary depending upon the number of hydroxyl groups and the presence or absence of (–CHO) or (=CO) groups.

Plants take up nitrogen from soil either in the form of nitrate or ammonia. Nitrate taken by plants is reduced by nitrate reductase and nitrite reductase enzymes. They catalyze step-wise reduction of nitrate to nitrite and nitrite to ammonia. Ammonia occupies the key position in nitrogen metabolism for the synthesis of organic nitrogen. Glutamic acid dehydrogenase, GOGAT and GS are important enzymes which are involved in the conversion of ammonia into glutamic acid, a primary amino acid. The ?-amino group of several amino acids is formed as a result of transamination reaction through transaminase/ aminotransferase enzymes. This process is of great significance in amino acid metabolism.

1. Protein Estimation

The method developed by Lowry et al. (1951) is largely followed for the estimation of soluble protein. This method is quite sensitive and gives a moderately constant value.

Protein may also be estimated by analyzing the total nitrogen content. Multiplying total nitrogen value with 6.25 will give the crude protein content. However, the nitrogen per cent varies with amino acid composition of proteins. For more refined expression of protein percentage, different factors are used for different samples. Some of the conversion factors for cereal and oilseed are given below:

1. Deoxyribo Nucleic Acid (DNA)

DNA is a genetic material, originally confined to nucleus of a cell. In plants, some DNAs are also present in mitochondria and chloroplasts. DNAs are vital macro molecules having high molecular weight.

Isolation of plant DNA is cumbersome or difficult primarily because of the presence of a number of secondary plant products. Secondly, a protocol, which is found suitable for isolation of DNA in one plant, may not work in other. The efficiency of DNA extraction depends on many factors, such as sample material, ionic conditions of the extraction medium and type of lysing agent used etc.

There are two methods, one developed by Dellaporta et al. (1983) and the other by Murray and Thompson (1980) which are most commonly used for extraction of DNA in a number of plant genera.

Murray and Thompson’s (1980) method is described here below with minor modifications.

Fatty acid esters of glycerol are known as fats or lipids. A fat, in its liquid state, is called ‘oil’. Oilseed plants such as castor, groundnut, sunflower, safflower, linseed etc. contain oil as reserve food in their seeds.

It must be noted that a small quantity of free fatty acid is usually present in oils along with the triglycerides. It increases during storage. The free fatty acid content of an oil is called as ‘acid number’ or ‘acid value’. The keeping quality of oil relies upon the free fatty acid content.

During germination of oilseeds, lipases play an important role in hydrolyzing the stored oils to fatty acid and glycerol. Fatty acid is metabolized to acetyl-COA, which may take part in glyoxylate cycle forming sugars from fatty acid involving the enzymes- Isocitritase and Malate Synthetase.

Photosynthesis is a very important parameter in plant physiological studies. It is the only process in nature which utilizes solar energy and converts it into chemical energy. It has a direct bearing on plant productivity. By understanding the process of photosynthesis, manipulation of productivity can be made possible.

1. Measurement of Photosynthesis Rate

Measurement of CO₂ uptake provides an alternate and direct method of measuring productivity, with important advantages over measurements of change in dry weight. It is instantaneous and non-destructive.

Infra Red Gas Analyzer (IRGA) is an equipment which is most widely used for the measurement of photosynthesis in situ in the field or in detached leaves under laboratory conditions. It is less time consuming and reliable. It can also be used for the measurement of rate of respiration and CO₂ compensation concentration (CO₂ CC), provided a little alteration is made in the chamber.

Respiration rate, irrespective of plant parts (viz. leaves, fruits or seeds), is considered to be an important parameter in plant physiological studies. Respiratory gas exchange is just reverse or opposite to that of photosynthesis and as the later is an energy building phenomenon where ATP is stored in the photosynthates produced; respiration involves breakdown of photosynthates for release of energy to be utilized for the performance of other activities of life. Respiration plays an important role during seed germination. A germinating seed requires a lot of energy which is derived from the breakdown of different types of food reserved in the cotyledons. On the basis of respiration a climacteric fruit can well be distinguished from a non climacteric one; as the former reveals a characteristic ‘Climacteric Burst’ in respiration, which is completely lacking in the later.

There are a few instruments used for the measurement of respiration in plants viz.:

Free radicals, such as singlet oxygen (¹_O2), superoxide (O₂ ^.–) and some active derivatives of oxygen, like H₂O₂ are although generated in plant cells during the course of normal metabolic processes, but their production is notably enhanced when the plants are exposed to any kind of stress environment. The reactive oxygen species damage important cellular components, including cell membrane by peroxidation of lipid and de-esterification of fatty acids.

Plants have evolved a wide range of mechanisms to contend with this problem. The antioxidant defence system of a plant comprises of a variety of antioxidant molecules and enzymes which take care of free radicals produced in the cells. These enzymes are popularly termed as the ‘Scavenging Enzymes’ or ‘Antioxidative Enzymes’.

Conversion of oxygen molecules into its reactive form leads to induction of a sort of stress in plants, to which we call as ‘Oxidative Stress’. It is the equilibrium between oxidative stress and antioxidant enzymes, which determines the fate of a plant.

Plants possess scavenging enzyme system to protect them from destructive oxidative reactions. These enzymes are not restricted to the intercellular compartment but are also found in the apoplast to a limited extent. Some of the enzymes are as follows:-

There is loosening of cell wall of climacteric fruits during ripening. The enzyme pectin methyl esterase (PME), is responsible for the de-esterification of pectin (major constituent of cell wall) into pectic acid (or Polygalacturonic acid) and methanol. Polygalacturonic acid serves as a substrate for PG (Polygalacturonase) enzyme. This enzyme (PG) starts depolymerization of pectic acid leading to the formation of small chains of tri, di and monogalacturonic acids.

A large number of pathogens also have been reported to produce pectic enzymes to macerate the plant tissue. Hence, in addition to its role in ripening process and shelf life of fruits, the enzyme PG is also studied in relation to the diseases of the plants.

Plant hormones and growth regulators are compounds that influence flowering, aging, root growth, prevention or promotion of stem elongation, prevention of leaf emergence and/or leaf fall and many other physiological processes. These substances produce major growth changes at a very low concentration.

Hormones are endogenously produced substances in plants while plant growth regulators may be synthetic compounds (i.e. IBA and Cycocel) that mimic naturally occurring plant hormones or they may be natural hormones that are extracted from plant tissue (e.g. IAA).

These growth regulating substances, most often, are applied as a spray to foliage or as a liquid drench to soil around a plant’s base. Generally, their effects are short-lived and they may need to be re-applied in order to achieve the desired effect.

Plant growth regulating substances are normally classified into five groups viz.: (i) Auxins, (ii) Gibberellins; (iii) Cytokinins; (iv) Ethylene; and (v) Abscisic acid.

For the most part, each group contains both naturally occurring hormones as well as synthetic substances. Physiological functions of each and every group of growth regulating substances are given below in brief:

Ascorbic acid (vitamin C) is present in almost all fresh fruits and vegetables in varying quantities ranging from 0.02 to 1.0 mg per g fresh weight. It is most abundantly found in bittergourd and berries.

The method for estimation of ascorbic acid is given here below as described by Albrecht (1993). It is an easy and simple method based on titration technique.

Phenols constitute a class of aromatic organic compounds having at least one hydroxyl group attached directly to the benzene ring.

A Flame Photometer has long been in use for analysis of elements; however, it is now being gradually replaced by Atomic Absorption Spectrophotometer. The reason being, that the former can analyze only a few elements such as sodium (Na), potassium (K), calcium (Ca), barium (Ba) and lithium (Li), whereas the later can be used to analyze almost all the elements known, provided a specific Hollow Cathode Lamp (HC), for a specific element, is available. In addition, Atomic Absorption Spectrophotometer is more precise and accurate in measurement as compared to Flame Photometer.

In this chapter, we shall deal with the operation techniques, working principles and a brief description of each and every component/constituent unit of a Flame Photometer as well as an Atomic Absorption Spectrophotometer.

1. Mitochondria

Mitochondria are known as the “Power House” of a cell because they are the site of ATP production. In addition, they are also involved with photorespiration and oxidation of fatty acids and work in association with peroxisomes, glyoxysomes and chloroplasts.

Isolation of intact mitochondria is rather difficult. This is because the cell walls of plant tissue are rigid as compared to the single-layered wall surrounding the mitochondria. A significant fraction of mitochondria suffer structural damage during the process of tissue homogenization. Therefore, the pelleted mitochondria need further purification by sucrose gradient centrifugation.

Mitochondria can be isolated from green as well as etiolated leaf; however, the latter is preferred to obtain higher yield of mitochondria (Moore and Proudlove, 1983).

In this chapter, we describe the methods of preparation of some of the buffers, most commonly used in the assay of enzymes required for plant physiological and histochemical studies.

The buffers have been arranged in an alphabetical order starting with their names. These methods are not necessarily identical with those of the original authors. It is advised that the users should re-determine the titration curves of majority of the buffers. These buffers have also been listed in Method in Enzymology, Vol. 1 (1955), pp. 138-146.

1.1 Importance of Food Process Engineering

Food occupies the first position among the hierarchal needs of human beings. It is more important than shelter and clothing. After production and be forec on sumpti on, food commodities are subjected to nume rous unfavourable physical, chemical, microbial or parasitic factors which may cause their spoilage or disease when consumed. To prevent such spoilage, a process engineer has to understand these and provide remedial measures. The two primary goals of processing operation are first to minimize post harvest losses and second value addition of raw food commodities through preservation and processing.

Present level of production of different types of food commodities and their estimated post-harvest losses along with monetary values are given in Table 1.1. It shows that various food items get lost, to the tune of 65 million tonnes, worth Rs. 76,000 crores per annum, during post-harvest phase. A substantial amount of these losses could be prevented if appropriate agro processing centers, having backward linkage with farmers to ensure constant supply of quality raw food materials, are established and operated. Profit generated through value addition must also be shared with farmers, who are only people in the world to create wealth every year, in the form of food, fiber and other commodities necessary for human survival. It can bring sea change in rural areas, where 65-70% population live, in respect of economy, health and happiness.

It sounds simple, but the above process is fairly complex and requires lot of coordination in order to succeed. The major challenge is to minimize the losses during the various post harvest operations and keep the food as fresh as possible. In order to do so, one must first try to understand the various causes of post harvest losses of food commodities and the various factors that influence theses causes. Secondly, we must use those food processing operations that will not only extend the shelf life of the produce but also convert the raw food commodities to more acceptable product to the consumer.

2.1 Introduction

Many complicated engineering principles are to be analyzed and applied, before any food processing operations takes shape of a commercially viable enterprise. The size and type of the required equipments have to be specified and their sequences have to be arranged. Further, the process analysis consists of understanding of material and energy balances. This chapter provides the application of steady state conservation of mass and energy equations to analyze food-processing operations.

3.1 Introduction

The subject of heat transfer is firmly grounded in the sciences of thermodynamics and fluid mechanics. It has become exceedingly important for engineers to possess a clear understanding of the principles of heat transfer and its application to a large number of problems. In view of process engineering, the knowledge of heat transfer and its application is most important, because heat transfer is one of the basic unit operations in the agricultural processing activities. The design of process equipments, refrigerators, boilers or type of heat exchangers, thermal treatment of food grains, cooling of perishables, pasteurizations, sterilizations, freezing of milk and fruit juices and a large number of similar applications involve consideration of heat transfer rates. There may be other situations where heat is being generated during a certain operation, and may be detrimental to the equipment and the food product. In such cases we are often faced with the task of removing the undesirable amount of heat as far as possible.

4.1 Introduction

The food preservation may be define as the state in which the food may retained over a period of time without being contaminated by pathogenic organism or chemicals and without losing its colour, texture, flavour and nutritional value. All the methods of food preservation must provide such an environment in and around the preserved food so that it produces one or more unfavourable conditions to the continued activity of the spoilage agents. The various methods used for food preservations are:

5.1 Introduction

The medium used in various unit operations of food process engineering such as heating, cooling, drying, mixing and transport processes, is moist air, which is a mixture of two substances. One of these is dry air which itself is a mixture of number of gases and the other is water vapour which may exist in a saturated or superheated state. The properties of moist air are called psychrometric properties and the subject, which involves determination of thermodynamic properties of moist air, is known as psychrometry. Thus, psychrometry is that branch of engineering science, which deals with the study of moist air i.e. dry air mixed with water vapour or humidity. Knowledge of properties of moist air is imperative in design of systems such as air- conditioning equipment for storage of fresh produce, dryers for drying of cereal grains, and cooling towers in food processing plants. In this chapter, important thermodynamic properties used in psychrometric computations are defined. Psychrometric charts useful in determining such properties are presented. In addition, various psychrometric process used in food process engineering are discussed.

6.1 Introduction

In general, the terms drying and dehydration are interpreted as the removal of moisture from a food product. Removal of moisture from a food product offers an excellent protection against most common causes of food spoilage. The drying or dehydration process achieves such protection by reducing the moisture content of the food product to levels which are adequate to limit microbial growth or other adverse chemical reactions. During drying, heat and mass transfer occur simultaneously (Figure 6.1). Heat is transferred from the bulk gas phase (i.e. hot air used for drying) to the solid phase (i.e. food product to be dried), where part of it is utilized for vaporization and the remainder is taken up by the solid phase as sensible heat. Mass (i.e. moisture of food product) is transferred in the form of vapour through various resistances, first from the interior of the solid to the surface, then through vaporization at or near the surface and finally in transport of the vapour into bulk gas phase. Dehydration is the removal of moisture close to bone dry condition.

7.1 Introduction

Evaporation is an unit operation which converts a liquid into a vapour, leaving behind a concentrated solution or a higher boiling liquid. In other words, the goal of evaporation is to vaporize most of the water from a solution containing a desired product. Heat is the main tool in evaporation, and the process occurs more readily at high temperature and low pressures. Removal of water from foods provides microbiological stability, reduces deteriorative chemical reactions, and reduces transportation and storage costs. Evaporation can be distinguished from some other drying methods in that the final product of evaporation is a concentrated liquid, not a solid. It also differs from distillation, since the vapours produced in the evaporator are not further divided into fractions.

It is well known that lowering the temperature of food stuffs reduces microbiological and biochemical spoilage and thus extending the shelf life of the product. In addition, it is generally agreed upon that the reaction rate is reduced by half by lowering the temperature by 10°C. Though low temperature preservation process has been recognized as a preservation technique for several hundred years, the major developments in its utilization have occurred only in the last century.

The various unit operations of food process engineering applied to solve the problem of changing the compositions of solutions, mixture and solids. Often these operations must be conducted on large scale to meet the demands of a large manufacturing plant. In food processes engineering these operations are often referred to as separation / concentration / extraction / purification process. Usually these operations are directed towards separating substances into its component parts or concentrating a particular substance from another.

10.1 Introduction

Milk is a unique substance in that it is both consumed, as fluid milk with minimal processing and it is the raw material used to manufacture a wide variety of products. From the nutrition point of view, milk is an almost ideal food and considered to be perfect food. The human body needs more than thirty distinct materials in its food supply. No single food stuff supplies all of them but milk supplies nearly all. Since milk by its nature is perishable and an ideal product for bacterial growth, so it must be carefully handled, both during production and while processing. This chapter focuses on the technological (processing) aspects of the transformation of milk into its various consumer products, including beverages, fermented products, concentrated and dried products, butter and ice cream.