Studies on Lactic Acid Bacteria from Tropical Fish and Shellfish

221 Pages·2011·11.86 MB·English

by  Seema Nair

STUDIES ON LACTIC ACID BACTERIA
FROM TROPICAL FISH AND SHELLFISH
'Ihesis submitted to
rrne Cochiti 'ilnioersitu ofScience ana rrecnno{ogy
Cocnin - 682 022
In partiai[utfillmen: of tne requirements for the deqree of
Doctor of PliiCosopliy
In
iMicrobioioqu
{Under the !Facu{ty ofMarine Sciences)
SEEMA NAIR P.
Microbiology, Fermentation and Biotechnology Division
Central Institute of Fisheries Technology, Matsyapuri P.D.
Kochi - 682 029
November 2000
CERTIFICATE
CENTRALINSTITUTEOFFISHERIES TECHNOLOGY
(IndianCouncil ofAgricultural research)
WillingdonIsland, Matsyapuri. P.O.,Cochin-682029.
Dr. P.K.Surendran,M.Sc.,Ph.D.,F.S.F.T., ARS,
Principal Scientist& HeadofDivision,
Microbiology, Fermentation& BiotechnologyDivision.
Thisisto certifythatthisthesisentitled "Studies on Lactic Acid Bacteria from
Tropical Fish and Shellfish "embodiedtheresultsofthe originalresearchworkdone
by Smt.Seema Nair, P under my supervision and guidance from 11- 10- 1995to
02-10-1999. I furthercertifythat nopartofthisthesishaspreviouslyformedthe basis of
theaward ofanyDegree,Diploma, Associate-ship, andFellowshipor other similar titles
of this or any other University or Societies. She has passed the Ph.D. Qualifying
ExaminationsoftheCochinUniversityofScienceandTechnologyinAugust,1999.
Cochin-682 029.
zo"
November, 2000. (Dr.P.K.Surendran)
OR.P.1tSURENDRAII
..........,.f_ '...
PRINCIPALSCIENTISTI teD~DMStON
&8........,
Contents
Page no
CflAPTER 1
INTI~ODUCTION 1
(~I-IAPTER 2
I~EVIEW OF I-JITE]~AT1JRE 3
2.1 Fish Bacteriology 3
2.1.1 Bacterialfloraof freshfishandshellfish intemperate region 4
2.1.1.1 Quantitative aspects 4
2.1.1.2Qualitativeaspects 5
2.1.2 Bacterialfloraoffreshfishesand shellfish intropical region 6
2.1.2.1 Quantitative aspects 6
2.1.2.2Qualitative aspects 8
2.1.3 Bacteriologyof frozenfishandshellfish 9
2.1.3.1 Quantitative aspects 9
2.1.3.2Qualitative aspects 10
2.2 Bacterial spoilage ofFish 11
~'.
"
2.3 Fish preservation 14
2.4 Pathogenic bacteria associated with fish 21
I
~in (.~I
2.5 seafoods 25
2.6 Lactic Acid Bacteria 27
2.6.1.1 Streptococcus 29
2.6.1.2 Lactococcus 30
2.6.1.3 Enterococcus 31
2.6.1.4Carnobacterium 32
2.6.1.5 Leuconostoc 33
2.6.1.6Pediococcus 34
2.6.1.7 Lactobacillus 34
2.6.2Lactobacillus fromfishandfisheryproducts 36
2.6.3 Lactobucillus as probiotics 38
2.6.4 Pathogenic Lacticacid bacteria 38
,2.6.5 Lacticacid bacteria inBiopreservation 39
2.7Bacteriocins 41
2.7.1 Bacteriocins ofLacticacid bacteria 41
2.7.1.1 BacteriocinsofLactococcus 41
2.7.1.2 BacteriocinsofPediococcus 42
2.7.1.3 BacteriocinsofLeuconostoc 43
2.7.1.4 BacteriocinsofCarnobacteria 43
2.7.1.5 Bacteriocins ofLactobacillus 44
2.7.2 Nonbacteriocin-inhibiting compounds 46
2.7.2.1 Diacetyl 46
2.7.2.2 Acetaldehyde 46
2.7.2.3 Hydrogenperoxide 46
CHAPTER3
MATERIALS AND METHODS 47
-'.1 Materials 47
3.1.1 Bacterialstrains 47
3.1.2 Fish 47
3.1.3 Media 54
1.1.4 Dehydrated media 54
3.1.5 Compounded media 54
3.1.4 Indicatorsolutions 60
3.1.5Testreagents 60
3.2 Methods 62
3.2.1 Samplingoffish/prawn forTotal Plate Count 62
3.2.1.1 Pourplatemethod 62
3.2.1.2Spreadplate method 62
3.2.2Enumeration ofLacticacid bacteria 63
3.2.3 Isolation ofLacticacid bacteria 63
3.2.4 Maintenance ofLacticacid bacteria 63
3.2.5 IdentificationofLacticacid bacteria 64
3.2.6 Factors influencingthe growth ofLacticacid bacteria 67
3.2.6.1 Effectoftemperature on growth 67
3.2.6.2 EffectofpJ1 on growth 67
3.2.6.3 E1Tect of Na(~1 ongrowth 67
3.2.6.4 Effectofglucoseconcentration onthe growth and
acidity produced in the medium 68
3.2.7 Titrable acidity 68
3.2.8 Wateractivity 68
3.2.9 Moisture content 68
3.2.10 Saltcontent 69
3.2.11 Detectionofantibacterial activity 69
3.2.12 Characterization ofantibacterial components produced by
Lacticacid bacteria 70
3.2.12.1 Organicacids 70
3.2.12.2 Hydrogenperoxide producxtion 71
3.2.13 Purificationofbacteriocins 71
3.2.14 Characterization ofbacteriocins 72
3.2.14.1 Heatsensitivity 72
3.2.14.2 Protease sensitivity 72
(~~IAPTER 4
I~F:S(JLTS AND DISCUSSION 73
4.1 Total bacterial count vis-a-vis Lactic acid bacterial count in fresh fish 73
4.1.1 Total bacterialcount infresh fish 73
4.1.2Lacticacid bacterialcount infresh fish 74
4.2Total bacterial countvis-a-vis Lactic acid bacterial count in fresh prawn 76
4.2.1 Total bacterialcount infresh prawn 76
4.2.2Lacticacid bacterialcount infresh prawn 77
4.3Total bacterial countvis-a-vis Lactic acid bacterial count in frozen fish 78
4.3.1 Total bacterialcount infrozenfish 78
4.3.2 Lacticacid bacterialcount infrozen fish 79
4.4Total bacterial countvis-a-vis Lactic acid bacterial count in frozen prawn 79
4.4.1 Total bacterialcount infrozenprawn 79
4.4.2 Lacticacid bacterialcount infrozenprawn 80
·4.5 Total bacterial countvis-a-vis moisture content, water activity
and saltcontent in cured fishes of commerce 80
4.6Total bacterial count in Ocean fresh fish 81
4.7 Total bacterial count vis-a-vis Lactic acid bacterial count in
farmed fish/prawn 82
4.8Total bacterial count in pickled fish/prawn 83
4.9 Studies on Lactic Acid Bacteria 84
4.9.1.1IsolationofLABfromfreshfish 84
4.9.1.2 Isolationof LABfromfreshprawn 84
4.9.1.3 Isolationof LABfromfrozenfish 84
4.9.1.4 IsolationofLABfromfrozenprawn 85
4.9.2Morphologicalcharacteristicsof LABcultures 85
4.9.3 Hornoferrnentative and heterofermentativenatureof LAB 86
4.10 Studies on Lactobaclllus isolated from fish and shellfish 88
.;l.11 Characterization of Lactobacillusspecies 89
4.11.1 Lplantarum 89
4.11.2 Lbrevis 90
4.1 1.3 Lidivergens 90
4.1 1.4 Lgasseri 90
4.11.5Lcaseissp rhamnosus 91
4.11.6Lfermentum 91
4.1 I.7L.viridescens 91
4.11.8 l.farciminis 92
~L11.9 L.buchneri 92
4.I1.10Lucidophilus 92
4.11.11 Lulimentarius 92
4.1 1.12 Lanimalis 93
4.11.13 L.rcutcri 93
4.12 Distribution ofLactobacillus in fresh and frozen fish and prawn 93
4.12.] Lactobacillus infresh fish 93
4.12.2Lactobacillus infresh prawn 93
4.12.3 Lactobacillus infrozenfish 94
4.12.4 Lactobacillus infrozen prawn 94
/
4.13lbe Biochemical and antibacterial investigations of
I
Lactobacillus cultures 96
4.13.1 Fermentationofsugars 96
4.13.1.1 Sugarfermentation byL.plantarum 97
4.13.1.2 Sugarfermentation byL.brevis 98
4.13.1.3 Sugarfermentation byLdivergens 98
4.13.1.4 Sugar fermentation byL.fermentum 98
4.13.2 Studies oneffectofglucose, NaCIand incubationtemperature
on growthand bacteriocin production byLactobacilluscultures 100
4.13.2.] Effectofglucoseconcentration on loweringofpH ofthe
I
culturemediabyLactobacilluscUlture~ 100
4.13.2.1.1 Titrable acidity byLactobacillusspeciesy 101
4.13.2.2 EffectofNaCI inculture mediaonthe growth of
Lactobacilluscultures 102
4.13.2.3 Effectof incubationtemperature on the growth of
Lactobacilluscultures 103
4.13.2.4 Effect ofpI-Iofthe media onthe growthof
Lactobacillus cultures 104
4.1 ).3 Studiesonantibacterial activityofLactobacilluscultures 104
4.13.3.1 ScreeningofLactobacilluscultures forantibacterial
activityagainst indicator/pathogenic bacteri(~~"':~" 104
4.]3.3.1.] InhibitionbyL.plantarum 105
4.13.3.1.2 InhibitionbyL.brevis 106
4.13.3.1.3 InhibitionbyL. divergens 106
4.13.3.1.4 InhibitionbyL. caseissp rhamnosu 106
4.13.3.1.5 Other Lactobacillusgroups 106
4.13.3.2 Nature ofantibacterial activity byLactobacillus 107
4.13.3.3 Characterization ofantibacterial activity by
Lactobacillus cultures 107
4.13.3.4 Antibacterial activityofbacteriocins against other
Lactobacilluscultures 109
4.13.3.5 Antibacterial activityoflacticacid andacetic acid 110
4.13.3.6 Antibacterial activitydue to Hydrogenperoxide 111
4.13.3.7 Antibacterial activitydueto bacteriocins 111
4.13.4 Antibacterial activity ofbacteriocin concentrates fromLactobacillus
cultures 112
4.13.4.1 Antibacterial activityofbacteriocinconcentrates against
pathogenic/indicator bacteria 112
4.13.4.1.1 Actionofbacteriocins against Listeria monocytogenes 112
4.13.4.1.2 Actionofbacteriocins against Bacillus cereus 112
4.13.4.1.3 ActionofbacteriocinsagainstStaphylococus aureus 113
4.13.4.1.4 ActionofbacteriocinsagainstSalmonella typhimurium 113
4.13.4.1.5 Action ofbacteriocins against Escherichia coli 113
4.13.4.1.6Action ofbacteriocins against Vibrio cholerae and
Vibrio vulnificus 114
4.13.5 Effectofsalt, pH, incubationtemperature and glucose
concentration onthe antibacterial activityofLactobacilluscultures 114
4.13.5.1. Effectofsalt 114
4.13.5.2. EffectofpH 115
4.13.5.3.Effect oftemperature ofincubation 115
4.13.5.4.Effect ofglucoseconcentration inthe culture media 116
(~l-ll\Pl-'ER 5
SllMMAI~Y AND C()NCLUSION 119
CHAPTER 6
REFERENCES 12~
Chapter.1
Im'(j(O(j)VCTIOW
1. Introduction
Lactic acid Bacteria (LAB) are characterized as Gram-positive, usually non-
motile, non-sporulating bacteria that produce lactic acid as a major or sole product of
fermentative metabolism. Members ofthis group contain both rods (Lactobacillus and
Carnobacterium) and cocci (Streptococcus, Leuconostoc, Pediococcus, Lactococcus,
Weissella, Oenococcus, Vagococcus and Tetragenococcus); they are generally catalase
,~
negative and lack cytochromes. LAB are nutritionally fastidious, requiring
carbohydrates, amino acids, peptides, nucleic acid derivatives and vitamins. The LAB
of importance in foods mainly belong to the genera: Lactobacillus, Lactococcus,
Leuconostoc, Oenococcus, Pediococcus and Streptococcus.
Lactic Acid Bacteria (LAB) have for centuries been responsible for the
fermentative preservation ofmany foods. They are used to retard spoilage and preserve
foods through natural fermentations. They have found commercial applications as
starter cultures in the dairy, baking, meat, fish, and vegetable and alcoholic beverage
industries. They are industrially important organisms recognized for their fermentative
abilityas well as their nutritional benefits. These organisms produce various compounds
such as organic acids, diacetyl, hydrogen peroxide and bacteriocins or bactericidal
proteinsduring lactic fermentations. Not only are these components desirable for their
effects on food flavour and texture, but they also inhibit undesirable microflora. LAB
andtheir products give fermented foods distinctive flavours, textures and aromas while
preventingspoilage, extending shelf-life and inhibiting pathogenic organisms. Currently,
there is some interest in the possible use of LAB and their metabolic products as
biocontrol agents, biopreservatives for non-fermented foods and probiotics.