Mikroba Termofilik dalam Proses Termal

10/24/16

Mikroba Termofilik dalam Proses Termal Ratih Dewanti-Hariyadi ter Dept. of Food Science and Technology Bogor Agricultural University

Outlines •  Bahaya dalam Produk Steril Komersial •  Pembusuk dalam Produk Steril Komersial •  Spora Bakteri •  Ketahanan Panas Spora Bakteri •  Illustration : Thermally Processed Dairy

Ra#h Dewan#-‐Hariyadi/2016

Ra*h Dewan*-‐Hariyadi/2016

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Bahaya dalam Produk Pangan Steril Komersial

• Bahaya utama : Clostridium botulinum • Bahaya lain yang mungkin adalah Bacillus cereus dan histamin • Spora C. botulinum dan B. cereus mungkin tersisa setelah proses termal • Histamin adalah bahaya yang berasal dari bahan baku ikan dari spesies scromboid yang mengalami penyalahgunaan suhu penyimpanan Ra#h Dewan#-‐Hariyadi/2016

Bahaya dalam Produk Pangan Steril Komersial C. botulinum

•  bakteri pembentuk spora,penghasil neurotoksin tumbuh pada pangan dengan pH, kandungan air dan zat gizi tertentu, tanpa oksigen. Lingkungan ini ada pada pangan berasam rendah yang diberi proses termal •  pengasaman sampai pH <4.6 menghambat germinasi spora secara sempurna •  pertumbuhan bacilli, kapang pada produk pangan asam atau diasamkan yang under-processed dapat meningkatkan pH sehingga dapat menyebabkanC. botulinum tumbuh dan membentuk toksin Ra#h Dewan#-‐Hariyadi/2016


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Bahaya dalam Produk Pangan Steril Komersial B. cereus & galur Bacilli lainnya

• Bakteri pembentuk spora • Bakteri menghasilkan toksin emetik & penyebab diarrhea • Bakteri ini lebih sensitif panas • B. cereus seharusnya bukan masalah penting pada pangan proses termal shelf-stable


Pembusuk dalam Produk Steril Komersial •  Mikroorganisme pembusuk pembentuk spora yang termotoleran : - lebih tahan panas dari C.botulinum - kebusukan pangan steril komersial berasam rendah: -  flatsour oleh spora termofilik aerobik (Bacillus stearothermophilus) -  pembusuk gas oleh spora termofilik anaerobik (Thermoanaerobacterium atau Clostridium thermosaccharolyticum) •  Mikroorganisme penyebab kerusakan sulfida : Desulfotomaculum (Clostridium) nigrificans, masalah bagi shelf-stable foods (sayur kaleng) yang didistribusikan pada suhu tinggi (tropis) Ra#h Dewan#-‐Hariyadi/2016


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Pembusuk dalam Produk Steril Komersial

•  Pembentuk spora asidofilik termotoleran juga

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dapat merusak makanan kaleng asam atau diasamkan (proses termal lebih ringan daripada makanan berasam rendah) : B. coagulans var thermoacidodurans, C.pasteurianum dan C.butyricum. Kontaminasi pasca proses oleh kelompok ini dan BAL : masalah pada pangan diasamkan hot-fill dan hold processes (tomat) Beberapa askospora kapang bertahan selama proses termal dan menyebabkan kebusukan pada buah kaleng. Byssochlamys spp. Talaromyces spp. dan Eupenicillium spp. toleran terhadap kandungan oksigen rendah


Bacterial Spores •  Bacterial spores are among the most resistant form of living organism •  Structure : multilayered spore coat composed of as many as 25 highly crosslinked polypeptides, thick layer of peptidoglycan, outer cortex, inner primordial cell wall



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Spora Bakteri


STRUKTUR ENDOSPORA BAKTERI

DNA MEMBRAN SITOPLASMA

KORTEKS

In# (core) SPORANGIUM

RIBOSOMA

SELUBUNG SPORA (SPORECOAT)



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1.  Proteinaceous coat :resist chemical &enzymes 2.  Cortex : specialized peptidoglycan, proper cortex formation is needed for dehydration of the spore core, which aids in resistance to high temperature

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3.  Cell wall : this layer of peptidoglycan will become the cell wall of the bacterium after the endospore germinates

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4.  Inner membrane : major permeability barrier against several damaging chemicals

5.  Core, exists in a very dehydrated state and houses the cell's DNA, ribosomes and large amounts of dipicolinic acid. Small acid-soluble proteins (SASPs) are also only found in endospores. These proteins tightly bind 5 and condense the DNA, and are in part responsible for resistance to UV light and DNA-damaging chemicals. . Other species-‐speciﬁc structures and chemicals associated with endospores include stalks, toxin crystals, or an addi9onal outer glycoprotein layer called the exosporium. 4


Endospore producing bacteria Bacillus : 32 – 69% GC (guanin-cytocin) •  B. cereus B. licheniformis •  B. maserans B. mesentericus •  B. subtilis B. megaterium •  B. coagulans B. anthracis •  B. natto Clostridium : 22 – 55% GC C. botulinum C. perfringens C. butyricum C. pasteurianus C. stearothermophilus C. saccharolyticum


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Endospore producing bacteria Desulfotomaculum : 38 – 52% GC •  Gram-positive, obligately anaerobic soil bacteria; •  a type of sulfate reducing bacteria à cause food spoilage in poorly processed canned foods à can be identified by the release of hydrogen sulfide gas à rotten egg smell Sporolactobacillus; Oscillospira; Sporohalobacter : (peranan ?)

Thermal resistance of spores Organisms B. sporothermodurans B. sporothermodurans G. stearothermophilus G. stearothermophilus B. cereus B. licheniformis Paenibacillus lac9s C. butyricum C butyricum

Source

Tempera ture (oC)

D-‐value

UHT milk Feed concentrate Ref. culture

100 100

160 min 468 min

121

42 sec 191 sec

Mas**s milk Raw milk Raw milk

100 100 100 85 (pH 7) 100 (pH 4.4)

6-‐8 min 103 min 64 min 23 min 10-‐15 min



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Example : Microorganisms in Milk •  Normal flora of udder : Staphilococci, streptococci, micrococci (50%), Corynebacterium, Escherichia coli

•  Aseptically drawn milk from healthy cow

generally contains hundreds – thousands (102 - 103 CFU) bacteria/ml

•  Poor milking practices, insufficient cleaning and disinfecton of the exterior of udder may influence the microflora of milk

Example : Microorganisms in Milk •  Milk from mastitic cow may have somatic cells •  • 


(e.g. polymorphonuclear/PMN and macrophages from blood cells) – screening test Milk from sick cow may have Mycobacterium bovis, Brucella abortus, Brucella melitensis, Brucella suis, Listeria monocytogenes, salmonellae or Coxiella burnetii (IDEAL) criteria for raw mik for heat processing Total Counts <105 Counts/mL Total somatic cells < 4.0x105 cells/ml

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Contaminant Microorganisms in Milk •  From fecal : salmonellae, Campylobacter jejuni, Yersinia enterocolitica, and E. coli

•  From soil : Bacillus •  From air : bacilli and clostridia •  From equipment : Pseudomonas, Alcaligenes, Flavobacterium, Chromobacterium; from milk stone matrix : thermoduric and thermoresistant bacteria micrococci, sporeformers, lactobacilli

•  Contamination from human workers Ra#h Dewan#-‐Hariyadi/2016

Raw Milk For Direct Consumption •  Raw milk may be sold directly on the farm or produced from certified and regularly monitored herds to be distributed locally at retail •  Many countries have stringent regulations that restrict or prohibit the retail sale of raw milk •  Raw milk is easily spoiled, refrigeration slow down the growth of most microorganisms except for psychrotrophs •  Psychrotrophs in raw milk : malty, rancid, yeasty, bitter, fruity, purid flavor and formation of purple (Chromobacterium spp) or red (Serratia spp) pigment sensory defect can be detected at106-107 CFU/ml ropiness due to Alcaligenes viscolactis Ra#h Dewan#-‐Hariyadi/2016


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Raw Milk For Direct Consumption • 

Before 1930 (application of pasterurization), milk was the major vehicle for foodborne outbreak associated with typhoid fever, diphtheria, septic sore throat, tuberculosis and brucellosis

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Various outbreaks and illness: - Coxiella burnetii, Brucella - Salmonella Dublin, S. Typhimurium, S. Thompson, S. Heidelberg, S. Enteritidis, S. Newport - Yersinia enterocolitica, Campylobacter. Listeria monocytogenes, Escherichia coli O157:H7 - hepatitis A, poliomyelitis FMD viruses - Staphylococcus aureus enterotoxins (15.6oC)


Thermal Processing of Milk •  Heat application batch or HTST pasteurization UHT sterilization •  In combination with : Mechanical treatment separation, centrifugation, homogenization filtration, agitation : churning, whipping of cream •  Also an application prior to : Water removal concentration, dehydration, ultrafiltration, condensed or powder milk, Ra#h Dewan#-‐Hariyadi/2016


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Thermoduric and Thermophilic Bacteria in Dairy •  Thermoduric bacteria are bacteria that survive pasteurization : - Bacillus - Geobacillus - Clostridium Recently - Paenibacterium - Anoxybacillus •  How do they survive heat pasteurization? -  Spore formers -  Biofilm formation Ra#h Dewan#-‐Hariyadi/2016

Nonsporeformer Thermoduric • Micrococcus •  Microbacterium •  Coryneform bacteria • Arthrobacter • Lactobacillus • Streptococcus • Enterococcus • Alcaligenes Ra#h Dewan#-‐Hariyadi/2016


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Control of Thermoduric sporeformers Control at farm level • Hygienic farm practices • Teat cleaning protocols • Equipment cleaning and sanitation Control in processing plant • Physical removal • Control biofilm formation • Inactivation Ra#h Dewan#-‐Hariyadi/2016

Thermophilic Bacteria in Milk •  Thermophiles microorganisms that tolerate and actively grow at high temperatures (>50°C) •  Thermodurics may or may not be thermophilic •  Thermophiles in dairy Anoxybacillus flavithermus Geobacillus spp Bacillus licheniformis Bacillus coagulans Bacillus subtilis Ra#h Dewan#-‐Hariyadi/2016


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Bacterial Biofilms •  Biofilm is a group of microorganisms adhering to solid surfaces and (generally) embedded in polysccharides matrices produced by the microorganisms •  Biofilm bacteria are more resistant to heat, sanitizing agents and (antibiotics) •  Biofilm helps thermoduric bacteria persist in the milk processing environment •  Biofilm is a source of contamination

Bioﬁlm of E. coli O157:H7 on SS surface (Dewan* and wong, 1995) Ra#h Dewan#-‐Hariyadi/2016

Pasteurized Milk Spoilage •  Off flavor : unclean, putrid, fruity average time for spoilage for HTST milk stored at 1.7oC 17 days); 5.6oC (12 days); at 10oC (6.9 days) HTST from well-operated plants do not spoil >10 days at 4oC Aseptically packaged milk last more than 10 days •  Less common : ropiness, partial coagulation •  Microorganisms responsible for spoilage : - surviving spore formers - thermoduric that forms biofilm : B. cereus and its lecithinase result in “sweet curdling” - psychrophilic in post process contamination : Pseudomonas, Flavobacterium, Chromobacter, Alcaligenes, Bacillus Ra#h Dewan#-‐Hariyadi/2016


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Pasteurized Milk Outbreaks •  Underprocessing Salmonella : S. Typhimurium; S. Braenderup Listeria monocytogenes (wet environmment) •  Post processing contamination Escherichia coli O157:H7, Yersinia enterocolitica •  Less frequently Staphylococcus enterotoxins M aflatoxin Ra#h Dewan#-‐Hariyadi/2016

Shelf stable milk •  Target kill step is to inactivate sporeformer bacteria (Bacillus and Clostridium) •  Spoilage UHT spoilage rate is 1/5,000 – 1/10,000 due to post processing contamination, sealing deficiency, pinhole, contamination during closure Bacillus and Pseudomonas may be killed but the enzymes may withstand heat, cause gelation Bacillus sporothermodurans spoilage (non pathogen) •  No outbreak due to UHT milk has been reported Ra#h Dewan#-‐Hariyadi/2016


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Cream •  Fat-rich fraction of milk centrifuged and separated by churning or other means, then pasteurized or sterilized •  Microbial quality of raw cream depends on the quality of milk •  Cream is a more sensitive product than milk, because it is common to stay in open boxes for longer period •  Spoilage due to thermophilic is common (B cereus), also from recontamination and from lipolytic Pseudomonas spp •  High fat content serve as protective agent for pathogens, several outbreaks recorded are due to Salmonella and Staphylococcus aureus enterotoxins Ra#h Dewan#-‐Hariyadi/2016

Thank You



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Mikroba Termofilik dalam Proses Termal

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