S P R I N G E R P R O T O C O L S H A N D B O O K S

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Springer Protocols Handbooks collects a diverse range of step-by-step laboratory methodsand protocols from across the life and biomedical sciences. Each protocol is provided in theSpringer Protocol format: readily-reproducible in a step-by-step fashion. Each protocolopens with an introductory overview, a list of the materials and reagents needed to completethe experiment, and is followed by a detailed procedure supported by a helpful notes sectionoffering tips and tricks of the trade as well as troubleshooting advice. With a focus on largecomprehensive protocol collections and an international authorship, Springer ProtocolsHandbooks are a valuable addition to the laboratory.

Basic Techniquesin Biochemistry, Microbiology

and Molecular Biology

Principles and Techniques

Aakanchha Jain

Bhagyoday Tirth Pharmacy College, Sagar, Madhya Pradesh, India

Richa Jain

Centre for Scientific Research and Development, People’s University, Bhopal, Madhya Pradesh, India

Sourabh Jain

Sagar Institute of Pharmaceutical Sciences, Sagar, Madhya Pradesh, India

Aakanchha JainBhagyoday Tirth Pharmacy CollegeSagar, Madhya Pradesh, India

Richa JainCentre for Scientific Research and DevelopmentPeople’s UniversityBhopal, Madhya Pradesh, India

Sourabh JainSagar Institute of Pharmaceutical SciencesSagar, Madhya Pradesh, India

ISSN 1949-2448 ISSN 1949-2456 (electronic)Springer Protocols HandbooksISBN 978-1-4939-9860-9 ISBN 978-1-4939-9861-6 (eBook)https://doi.org/10.1007/978-1-4939-9861-6

© Springer Science+Business Media, LLC, part of Springer Nature 2020This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of SpringerNature.The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

DEDICATED TO

MY MENTOR: MY FATHER

“Late Prof. P.C. Jain” and My family

Preface

In today’s era of integrated science, the boundaries of different subjects have been dissolved,and the students are merger of all biological sciences. Over centuries, the world of biologicalsciences has changed radically. Years back, there were no machines like PCR, MALDI-MS,PET, etc. and nearly no plant and animal strains of altered genetic codes, but the presentscenario announces new proteins and enzymes every week. With the increasing molecularbiology, the basic biochemical, microbiological, and biotechnological analysis needs to beadvanced appreciably. Herein, the book Basic Techniques in Biochemistry, Microbiology andMolecular Biology will cover experiments in microbiology and molecular biology (for BScand MSc students), while Section 2 will comprise of the experiments in biochemistry (as perrevised new syllabus RGPV and PCI). The present book will provide insight to practicals ofbiochemistry, microbiology, and molecular biology to build the practical base of subject forreaders. Thus, the book will be useful for both undergraduate and postgraduate pharmacyand life science students.

The book will cover applied aspects of practical described herein in addition to theprinciples of experiment and procedures. The protocols included are brief and clearlydefined. Appendices incorporate composition and preparation of media and buffers. Thebook will also apprise the students with the importance of the subject and acquaint them.The simplified experimental protocols chosen herein will be replicable in college, institute,and university laboratories.

We hope that this will enlighten and instigate the students of all Indian universitypharmacy colleges and that the book proposal has vim and vigor to allow its acceptancefor publication by Springer Nature.

We welcome suggestions and criticisms from all scientific communities.

Sagar, Madhya Pradesh, India Aakanchha JainBhopal, Madhya Pradesh, India Richa JainSagar, Madhya Pradesh, India Sourabh Jain

vii

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiAbout the Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

PART I INSTRUMENTS

1 BOD (Biochemical Oxygen Demand or Biological Oxygen Demand)Incubator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2 Laminar Air Flow/Biosafety Cabinets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3 Aseptic Hood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 Autoclave . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5 Hot Air Oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

6 Deep Freezer (�20 �C) (Low-Temperature Cabinet) . . . . . . . . . . . . . . . . . . . . . . . . 13

7 Refrigerator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

8 Compound Microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

9 Digital Colony Counter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

10 Digital Turbidity Meter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

11 Digital Nephelometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

12 Digital PhotoColorimeter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

13 Digital UV/Visible Spectrophotometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

14 Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

15 ELISA Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

16 Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

PART II MOLECULAR BIOLOGY

17 Enzyme Assay: Qualitative and Quantitative. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Qualitative Enzyme Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Quantitative Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

18 Antimicrobial Sensitivity Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

19 Extraction of DNA from Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 From Onion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 From Spinach and Cauliflower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

ix

20 Extraction of DNA from Animal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611 From Blood [Using Phenol Extraction Method] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612 From Blood [Using Isopropanol Precipitation] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623 From Mouse Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 From Pig Spleen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

21 Extraction of DNA and RNA from Microorganism . . . . . . . . . . . . . . . . . . . . . . . . . 651 From Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652 From Yeast Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

22 Estimation of DNA by Diphenylamine Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

23 Estimation of RNA Using Orcinol Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

24 Determination of Melting Temperature of DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

25 Quantitative Estimation of DNA and RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

26 Purification and Bioassay of Interleukin-1 and Interleukin-2(IL-1 and IL-2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

27 Quantitative Determination of TNF Alpha and Interleukin-6 . . . . . . . . . . . . . . . . 87

PART III MICROBIOLOGY

28 Sterilization of Glassware; Preparation and Sterilization of Media . . . . . . . . . . . . . 93

29 Sub-culturing of Bacteria, Fungi and Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . 101

30 Preservation of Microorganisms: Stabs, Slants, Lyophilizationand Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

31 Staining Methods – Simple Staining, Negative Staining, Gram’s Stainingand Acid-Fast Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

32 Isolation of Coprophilous Fungi (Moist Chamber Method). . . . . . . . . . . . . . . . . . 117

33 Isolation of Microorganisms from Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

34 Motility Testing – Hanging Drop Method and Stab. . . . . . . . . . . . . . . . . . . . . . . . . 121

35 Sterility Testing of Pharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1231 Method B: Direct Inoculation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

36 Physical, Chemical and Bacteriological Analysis of Water. . . . . . . . . . . . . . . . . . . . . 131

37 To Perform Biochemical Identification of Microorganism:IMViC (Oxidative Fermentation and Carbohydrate Source Utilization) . . . . . . . 141

38 To Perform Biochemical Identification of Microorganism by NitrogenSource Utilization or Urease Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

39 Immobilization of Enzymes and Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 1471 Enzyme Immobilization in Polyacrylamide Gel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472 Enzyme Immobilization in Alginate Gel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483 Enzyme Immobilization in Gelatin Gel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1494 Cell Immobilization in Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505 Immobilization of Microbial Cells in Gelatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

x Contents

PART IV BIOCHEMISTRY

40 Qualitative Systematic Analysis of Carbohydrates (Glucose, Fructose,Lactose, Maltose, Sucrose and Starch) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1551 Molisch’s Test (α-Naphthol Reaction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1552 Benedict’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1573 Fehling’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1584 Iodine Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1585 Nylander’s Test (Bismuth Reduction Test) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596 Cole’s Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607 Reduction of Methylene Blue Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608 Barfoed’s Test: Reducing Monosaccharide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1619 Bial’s Test for Pentose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16210 Seliwanoff’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16211 Hydrolysis of Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16312 Methylamine Test for Lactose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16313 Osazone Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

41 Protein Analysis in Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1671 Kjeldahl Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1682 Dumas (Nitrogen Combustion) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693 Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1704 Colorimetric Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705 Dye-Binding Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1715.1 Anionic Dye-Binding Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1715.2 Bradford Dye-Binding Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

6 Copper Ion-Based Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1726.1 Biuret Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1726.2 Lowry Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736.3 Bicinchoninic Acid Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

7 Ultraviolet Absorption at 280 nm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1748 Peptide Measurement at 190–220 nm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

42 Identification Tests for Proteins (Casein and Albumin) . . . . . . . . . . . . . . . . . . . . . . 177

43 Quantitative Analysis of Reducing Sugars by 3, 5-DinitrosalicylicAcid (DNSA Method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

44 Quantitative Analysis of Proteins by Various Methods Including Biuret. . . . . . . . 1851 Other Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1861.1 Bradford Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1861.2 Lowry Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1871.3 Protein Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

45 Qualitative Analysis of Urine for Abnormal Constituents . . . . . . . . . . . . . . . . . . . . 1911 Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1912 Biochemical Characterization of Urine to Detect Abnormal Constituents . . . . . . . 1933 Benedict’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1934 Tauber’s Test for Detection of Aldopentoses in Urine Samples. . . . . . . . . . . . . . . . . 1945 Bial’s Test for Detection of Pentoses in Urine Samples . . . . . . . . . . . . . . . . . . . . . . . . 1946 Seliwanoff’s Test for Detection of D-Fructose in Urine Samples . . . . . . . . . . . . . . . 1957 Mucic Acid Test for Analysis of Lactose or Galactose. . . . . . . . . . . . . . . . . . . . . . . . . . 1958 Detection of Acetone Bodies Using Rothera’s Nitroprusside Test . . . . . . . . . . . . . . 1959 Estimation of Acetoacetic Acid by Gerhardt’s Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Contents xi

10 Analysis of Proteins in Urine Sample by Sulfosalicylic Acid Test . . . . . . . . . . . . . . . 19611 Analysis of Proteins in Urine Sample by Bence Jones Protein . . . . . . . . . . . . . . . . . 19712 Determination of Bile Pigments by Harrison Spot Test . . . . . . . . . . . . . . . . . . . . . . 19713 Schlesinger’s Test for Urobilinogen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19814 Detection of Bile Salts in Urine by Pettenkofer’s Test. . . . . . . . . . . . . . . . . . . . . . . . 19915 Detection of Occult Blood in Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

46 Determination of Blood Creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2011 Serum Creatinine Test (Jaffe’s Test). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

47 Determination of Blood Sugar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2051 Folin-Wu Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2062 Glucose Oxidase Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

48 To Perform Oral Glucose Tolerance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

49 Determination of Serum Total Cholesterol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2131 Enzymatic Colorimetric Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2132 Sackett’s Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2143 Zak’s Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

50 Preparation of Buffer Solution and Measurement of pH . . . . . . . . . . . . . . . . . . . . . 217

51 Study of Enzymatic Hydrolysis of Starch. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

52 Determination of Salivary Amylase Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

53 Study the Effect of Temperature on Salivary Amylase Activity . . . . . . . . . . . . . . . . 227

54 Study the Effect of Substrate Concentration on Salivary Amylase Activity . . . . . . 231

55 Analysis of Butter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

56 Analysis of Milk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

57 Thin Layer Chromatography of Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

58 Thin Layer Chromatography of Amino Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

59 Paper Chromatography of Amino Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

60 Paper Chromatography of Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

61 Fat Characterization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2651 Methods for Characterization of Fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2651.1 Melting Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2661.2 Iodine Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2661.3 Saponification Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2671.4 Peroxide Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

2 Methods for Lipid Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2692.1 Fatty Acid Composition and Fatty Acid Methyl Esters (FAMEs) . . . . . . . . . . . 2702.2 Cholesterol and Phytosterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

62 Analysis of Bread. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Appendices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

xii Contents

About the Authors

AAKANCHHA JAIN is an Associate Professor at Bhagyoday Tirth Pharmacy College, Sagar,India. She possesses a throughout first class career. She has to her credit 11 national awardsincluding Nagarjuna Award 2010, Gour Samman 2010, Ranbaxy Science Scholar2012, and MP Young Scientist Award 2018. She is a Zealous Teacher and Researcher.She has published 23 review and research articles in peer-reviewed high-indexed journals.She was formerly INSPIRE DST Research Fellow at the Department of PharmaceuticalSciences, Dr. H. S. Gour University, Sagar, India, from where she has completed her PhD,MPharm, and BPharm. She has also published two invited book chapters (Wiley, PanStanford) covering wide aspects of biotechnology demonstrating her innovation andcreativity in delivering lasting results in the field. She has examined many aspects of drugdelivery through nanoparticles in her MPharm and PhD. Her main contributions have beenin the field of formulation and characterization of novel drug delivery systems, transdermaldrug delivery, and anticancer drug delivery systems.

RICHA JAIN is a Senior Scientist, working in the Centre for Scientific Research andDevelopment, People’s University, Bhopal, India, possesses more than 14 years ofresearch experience in the area of molecular biology, animal cell culture, microbiology,bioremediation, and industrial and therapeutic enzymes. Presently, she is working on twoprojects: (a) therapeutic enzymes arginases (anticancer) and (b) bioremediation of plasticwaste. She has published 21 research papers with 81 citations in research journals of highrepute. Among several feathers on her cap includes Best Paper Award for the presentationof her paper on “Bioinformatics: Importance of Proteomics and Genomics” during theNational Conference on “New Frontiers of Sciences” National Science Day Celebrations2003, Dr. Hari Singh Gour University, Sagar, 28 February 2003;All India Young ScientistAward for the presentation of her paper “Production of Proteins and Amino Acids UsingPoultry Feathers from a Protease Producing Strain of Streptomyces sp. CFS 1068” duringBhartiya Vigyan Sammelan 2007 in Silver Jubilee of All India Young Scientist Conference,M.P. Council of Science and Technology, Bhopal, 23–25 November 2007; 1st Prize for herpaper presentation on “Fluconazole-Loaded Cubosome: An Excellent Nanoparticle forTreatment of Cutaneous Candidiasis” during the National Seminar on RecentAdvancement in Drug Delivery Techniques and Drug Designing, School of PharmacyResearch, Peoples University, Bhopal, 26–27 September 2014; and Appreciation forOral Presentation in the International Conference on Translational Medicine in theTwenty-First-Century “Stem Cell Transplantation: Current Status,” BarkatullahUniversity, Bhopal, 11–14 April 2015. She has also presented paper entitled “Study ofAnti-adenocarcinoma Activity of Arginase by Streptomyces Species.” She is also a Memberof different societies like the Association of Microbiologists of India, Indian ScienceCongress Association, Institutional Biosafety Committee, etc. She has actively participatedin more than 30 scientific workshops and conferences and has also delivered invited guestlectures in workshops and conferences along with providing hands-on training in eightworkshops.

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SOURABH JAIN is an Associate Professor at Sagar Institute of Pharmaceutical Sciences, Sagar,India. He has completed his PhD from Shobhit University, Meerut, UP, India; MPharmfrom Smriti College of Pharmaceutical Education, Indore, MP, India; and BPharm fromB.R. Nahata College of Pharmacy, Mandsaur, MP, India. He is having 8 years of experiencein academic and research. He has published 24 papers in national and international journalsand conferences. His key research interests include standardization of Ayurvedic andpolyherbal formulation, antidiabetic screening and phytochemical isolation of medicalplant, and biochemical testing.

xiv About the Authors