APPLIED MICROBIOLOGY: FERMENTATION and



MICROBIOLOGY OF FOODS & C0SMETICS







I. FOOD MICROBIOLOGY



REF.: PHK - CHAPTER 43 (ON RESERVE); Tortora et al. Chap. 28; Madigan et al. Chap. 112 & part of Chap. 23

Hurst, A. and D. L. Collins-Thompson. 1979. Food as a Bacterial Habitat. pg, 79-134. In, Advances in Microbial Ecology, Vol 3., ed. M. Alexander. Plenum, New York.

Ryan, M. P., M. C. Rea, C. Hill, and R. P. Ross. 1996. An Application in Cheddar Cheese Manufacture for a Strain of Lactococcus lactis Producing a Novel Broad-Spectrum Bacteriocin, Lacticin 3147. Appl. Environ. Microbiol. 62: 612-619.


Simon, M. C., D. I. Gray, and N. Cook. 1996. DNA Extraction and PCR Methods for the Detection of Listeria monocytogenes in Cold-Smoked Salmon. Appl. Environ. Microbiol. 62: 822-824.


Broome, C. V. 1993. Listeriosis: Can We Prevent It? ASM News 59: 444-446.

 

Laberge, I., A. Ibrahim, J. R. Barta, and M. W. Griffiths. 1996. Detection of Cryptosporidium parvum in Raw Milk by PCR and Oligonucleotide Probe Hybridization. App. Environ. Microbiol. 62: 3259-3264.


Venkateswaran, K. T. Kurusu, M. Satake, and S. Shinoda. 1996. Comparison of a Fluoregenic Assay with a Conventional Method for Rapid Detection of Vibrio parahemolyticus in Seafoods. App. Environ. Microbiol. 62: 3516-3520.


Gahan, C. G. M., B. O'Driscoll, and C. Hill. 1996. Acid Adaptation of Listeria monocytogenes Can Enhance Survival in Acidic Food and during Milk Fermentation. App. Environ. Microbiol. 62: 3128-3132.


Acheson, D. W. K. and G. T. Keusch. 1996. Which Shiga Toxin-Producing Types of E. coli Are Important? ASM News 62: 302-306


Busscher, H. J., M. van der Kuijl-Booij, and H. C. van der Mei. 1996. Biosurfactants from thermophilic dairy streptococci and their potential role in the fouling of heat exchanger plates. J. Ind. Microbiol. 16: 15-21

McAuliffe, M. P. Ryan, R. P. Ross, C. Hill, P. Breeuwer, and T. Abee. 1998. Lacticin 3147, a Broad-Spectrum Bacteriocin Which Selectively Dissipates the Membrane Potential. Appl. Environ. Microbiol. 64: 439-445.

Tsai, G.-J. and M. A. Cousin. 1993. Partial Purification and Characterization of Mold Antigens Commonly Found in Foods. Appl. Environ. Microbiol. 59: 2563-2571.

Dieuleveux, V., D. Van der Pyl, J. Chataud, and M. Gueguen. 1998. Purification and Characterization of Anti-Listeria Compounds Produced by Geotrichum candidum. Appl. Environ. Microbiol. 64: 800-803.

Lampel, K. A., P. A. Orlandi, and L. Kornegay. 2000. Improved template preparation for PCR-based assays of detection of food-borne bacterial pathogens. Appl. Environ. Microbiol. 66: 4539-4542.

Cimons, M. 2000. Rapid foodborne pathogen ID system is making a difference. ASM News 66: 617-619.

Perl, P. 2000. Poison package. The Washington Post Magazine Jan. 16, 2000.


Doores, S. 1999. Food Safety Current Status and Future Needs. A report for American Academy of Microbiology. ON RESERVE


Lisle, J. T., B. H. Pyle, and G. A. McFeters. 1999. The use of multiple indices of physiological activity to assess viability in chlorine disinfected Escherichia coli O157:H7.

Garcํa, C., A. Martin, M. L. Tim๓n, and J. J. C๓rdoba. 2000. Microbial populations and volatile compounds in the 'bone taint' spoilage of dry cured ham. Letts. Appl. Microbiol. 30: 61-66.

Sparringa, R. A. and J. D. Owens. 1999. Inhibition of the tempe mold, Rhizopus oligosporus, by ammonia. 1999. Letts. Appl. Microbiol. 29: 93-96.




A. Food Spoilage and Preservation


1. Extrinsic and Intrinsic Factors - Figure 43.1 (PHK)



2. INTRINSIC FACTORS: pH, aw, water availability, natural anti-microbials, composition of the food, redox potential, physical nature - ground vs whole (WHY?)



concepts of osmophiles, xerophilic, halophilic, etc.







3.  Differences in Spoilage Based on Intrinsic Properties



4.  Natural antimicrobials: aldehydes and phenolics in cinnamon, mustard, oregano; other alkaloids, etc. in onion, garlic; eggs & lysozyme; did I mention that aflatoxin, amatoxins, mycotoxins are alkaloids?





5. EXTRINSIC FACTORS: temperature of storage, moisture conditions of storage



Figure 10. Hurst and Collins-Thompson





6. Preservation of Foods - Tables 43.3 and 43.4 in Text



a. Shrimp - use of bacteriocins from lactobacilli to preserve brined shrimp - OVERHEAD



b. pediocins - OVERHEAD



c. lacticin 3147 - OVERHEAD - use in controlling nonstarter lactic acid bacteria; now know (1998 article) mode of action is to interfere with membrane potential



d. other naturally produced bacterial inhibitors - phenyllactic acid and indollactic acid - OVERHEAD





B. The Major Microbial Components of Foods - Table 7 (Hurst & Collins-Thompson)



www.fightbac.org

http://vm.cfsan.fda.gov/list



Two web sites relating to the topic of food safety, poisoning, cosmetic safety



1. Enterobacteriaceae



Important genera are: Escherichia, Citrobacter, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Proteus and Yersinia.



The Escherichia, Enterobacter, Klebsiella, and Citrobacter are coliforms and important in the indication of sanitation as well as being indicator organisms of potential pathogens. Cheese however often has high coliforms but no pathogens (104/gm curd). Coliforms can't compete well at colder temperatures and pH below 5.5, but Klebsiella, Enterobacter & Citrobacter do grow well in milk in the refrigerator.



Salmonella sp. problems in the poultry industry; also can have viruses but most viruses are species specific.



Yersinia enterocolitica is a human pathogen and has been isolated from vacuum packaged meats suggesting that it grows well at <5 C.



Serratia sp. and Proteus sp. involved in spoilage of dry cured ham. OVERHEAD OF ISOLATES



Primary volatiles causing the odor: hexanal, 2-butanone, 5-butanone, ethanol, undecane, butanoic acid, dimethyl disulfide





2. Pseudomonads TABLE I FROM HURST & COLLINS-THOMPSON





MOST COMMON SPECIES: P. fragi, P. fluorescens, P. aeruginosa, P. tralucida, & P. ovalis. Many of these are facultative Psychrophiles; produce pigments, lipases, proteases at low temp. & are active at low temps. Also produce amines - trimethylamine from trimethylamine oxide.





3. Staphylococcus sp. and Micrococcus sp.



Staph grow as facultative and produce lactic acid from glucose; micrococci grow as obligate aerobes



Food poisoning: OVERHEAD:



salmonella, staph, clostridia & etc. TABLE: PHK



Staph aureus produces enterotoxins A, B, C1, and C2; requires about 106 cells/ gm of food for enterotoxin conc. to be high enough: salami, cheese, rich well aerated foods - tuna salad, mayonnaise; enterotoxin B not associated with growth but others are and B appears to be related to antibiotic resistance and is inhibited by fermentable carbohydrates. Source in humans: nose; animals - skin.



Do not survive storage well - unlike Salmonella typhimurium which could be cultured 1 yr after inoculation. Don't compete at all against gn and poorly against other gp.



Micrococci: often found but usually don't contribute to deterioration of food; occasionally found to cause "scented-sour" odor in packaged meat



4. Lactobacilli



Organisms included: Streptococcus, Lactobacillus, Leuconostoc, Pediococcus. General characteristics: gp, rods or cocci, produce lactic acid, nonmotile, nonsporeforming



Very good competitors in food: homofermentative - produce only lactic acid from pyruvate; require NADH and FADH; latter produces hydrogen peroxide (MIC to Staph is 4 ug/mL but with Lactobacillus lactis it is 125 ug/mL)



Probably imp. in Swiss cheese production in controlling clostridia when the oxygen conc. are low.



CO2 can prevent other organisms from growing; lactic are less sensitive



sequence is usually: mixed flora going to lactic cocci to lactobacilli: cheddar cheese - no lactic streps, 106-7 fecal strep/ gm & 108-9 lactobacilli/gm; Swiss cheese different since it starts at a high temp. fermentation by S. thermophilus and followed by thermophilic lactobacilli; sauerkraut Leuconostoc mesenteroides, L. plantarum, L. brevis. Also associated with yeasts and filamentous fungi is sourdough bred, yogurt; Roquefort cheese



More recently, dairy streptococci produce biosurfactant which stimulate detachment from heat exchangers and the biosurfactant has the effect also of leaving an ani-adhesive film on the heat exchangers. OVERHEAD - Fig 1. Busscher et al.



5. Spore-formers TABLE 2 HURST & COLLINS-THOMPSON



Bacillus cereus & clostridia







C. Food Borne Diseases - Table 43.5 (PHK)



"There is nothing so humiliating as to know what a controlling influence the intestines have on man's thoughts." John Luchansky.



1. Seriousness of problem





Example: Ball Park Franks and Sara Lee premium deli meats - 1 x106 / day



Refrigeration unit over conveyor belt with fluid drippping onto exposed meats. 11 of 12 samples positive for bacteria while previously only 3/25. Sanitation increased. First death in Oct. 19, 1998 of 74 yr. Old woman in Tenn.; next in Detroit 9520 in Nov.; Columbus (31) in Dec.; New York on Christmas day (75); pregnant mother survided but not her twins (Jan in Columbus); Total death toll - 21 in 22 states over 100 seriously injured in 22 states. Listeria monocytogenes. Recall then started in Jan 1999.



Est. 76 x 106 illness/yr. With 325,000 hospitilizations and about 5,000 deaths annually. Cost close to $30 billion



60% of cases not reported or underreported.



Now PulseNet for comparing with PCR fingerprints of outbreaks



OVERHEADS ON DISEASES - CAN BE FOUND IN Food Safety Reference on reserve in library.





2. Distinguish between an infection and an intoxication



a. Special problems with Listeria monocytogenes





3. Figures 3 & 4 Hurst and Collins-Thompson on clostridia





4. Listeriosis: meningoencephalitis, meningitis, stillbirths, neonatal infections



sources: precooked foods, soft cheeses, undercooked poultry (33% of patients with listeriosis also had foods in their refrigerators from which the same serotype of Listeria was isolated)



distribution: 2% dairy products, 20% soft cheeses and processed meats, 30% raw vegetables such as radishes, cabbage, 50% raw meat and poultry and pate.



problem: organism is cold tolerant (grows at 4 - 10 C) and salt tolerant; gram positive, nonspore-forming rod; hard to detect in food stuffs

solution to detection: PCR using extraction to remove PCR inhibitors in the food



prevention: OVERHEAD



5. Shiga Toxin Production by E. coli - Enterohemmoraghic strains



E. coil 0157:H7



Seriousness of the problem: over 30 outbreaks in last 2 years, over 100 strains involved; third most common cause of diarrhea after Campylobacter and Salmonella strains.



Normally, microbial load of stomach and upper intestine <104 organisms per mL; mid to lower 10 6 to 10 7 CFU/mL and lower >10 9 with E. coli d dominant component. Question is where organisms grow. In ileum or in colon.



Causes: infected cattle; dry fermented sausages, apple cider, milk, mayonnaise, water, cattle-person, etc.



Time Magazine





II. FERMENTATION AND FOOD PREPARATION





A. Milk Products - Table 43.6 (PHK)



1. Types of Milk



2. Yogurt: Lactobacillus bulgaricus and Streptococcus thermophilus in yogurt; the lactobacillus is proteolytic yielding amino acids which the streptococci consume (Biotechnol. Prog. 14: 963-965, 1998)



3. Cheeses - Table 43.7 (PHK)



OVERHEAD - CHEESE TREE



4. Production of Cottage Cheese



Pasteurize skim milk, cool to 70 F, add starter culture (L. lactis and L. cremoris and Leuconostoc sp. used for acid production by lactobacilli and diacetyl [flavor] by Leuconostoc), incubate for 12 hours



Get a firm curd, cut into cubes, cook at 125 F for >50 min



Whey is removed and stir; if want a dry curd cottage cheese to remove more whey



Salt added and mixed if want creamy style, preservative added, packaged & refrigerated



Need to have the Leuconostoc produce the diacetyl because if the lactobacilli do, they produce too much CO2 and the whey floats





5. Production of Cheddar Cheese



Whole milk is pasteurized, color (annato) is added, then the starter culture is added - same as above



Incubate at 86 F for acidity to increase by 0.2% (usually about 60 min), then rennet is added to destabilize the casein to produce paracasein which coagulates at the low pH and low temperature



Incubate for coagulation to occur, about 30 min.



Cooked at 100 F, whey drained, lactobacilli continue to degrade lactose, stirred (cheddaring) to loose whey and curd formed in a mat



Milled, salted, put into form, pressed for 16 hr to remove all whey



Dried for 5 days at 50 F, waxed, cured at 40 F for 2 - 12 months.



Sharp flavors due to breakdown of lipids, proteins in milk producing H2S, lactones, ketones, and thioesters. Some of this may be due to secondary microflora which gives non-Kraft cheeses their special tangs.



5. Biotechnological Aspects



Use of pure cultures



Rennin/Chymosin



Acidity control





B. Grain Products



1. Rice - OVERHEAD



2. Wheat/Barley/Hops - Fig 43.16 (PHK)



3. Wine - Fig. 43.16; malolactic fermentation occurs after alcohol production by lactic acid bacteria by converting malic acid to less acidic lactic acid; this fermentation also produces "aroma active compounds) (AEM 65:740-745, 1999).



REVIEW ARTICLE: Versari, A., G. P. Parpinello, and M. Cattaneo. 1999. Leuconostoc oenos and malolactic fermentation in wine: a review. J. Indust. Microbiol. Biotechnol. 23: 447-455.



Beside maleic acid, citric acid is metabolized to acetate and oxaloacetate. This latte is converted to pyruvate which goes to acetate, ethanol, lactic acid, diacetyl acetoin or 2,3-butanediol. Wine yeast also do the same thing. These account for the "complexity" of wines



4. Soybean - OVERHEAD









Tempe - soybean or pulse cake held together with Rhizobus oligosporus. When this is inhibited by increased pH (ammonia) get product deterioration.





C. Miscellaneous Other Fermented Foods - Table 43.8 (PHK)



1. Cabbage - OVERHEAD



2. Sausages - OVERHEAD



LeRoy, F. and L. de Vuyst. 1999. Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin Sakacin K. Appl. Environ. Microbiol. 65: 974-981.



3. The Chocoholics Dream Fermentation



Each cocoa pod contains about 40 seeds and weighs about 1 pound



The pods are cut by machete, the pulp/beans mixture (which contains 14% sugar and 1.5% pectin at pH 3.5) is scooped out and allowed to ferment for up to seven days during which time they are turned for aeration



The fermentation produces alcohols and acids from the sugar and heat both of which cause the cotyledons in the beans to undergo biochemical changes



The beans are sun dried, roasted at about 135 C, and broken into small pieces (kibbled)



The broken shells are blown away in a winnowing machine.



The nibs (small pieces) are reduced to a thick liquid (cocoa mass) which contains about 55% cocoa butter



About half of the cocoa butter is removed during pressing to make chocolate bars and the remainder is used for baking and beverages



Microbial succession (12 yeasts and over 30 species of bacteria identified):



Days 1-2 - yeasts + lactic acid bacteria



Days 2-3 - lactic and acetic acid bacteria



Days 4-7/8 - acetic acid bacteria and spore formers



Days 6-7/9 - fungi and spore formers



Biotechnological Needs



Which species of yeast/bacteria are really important - yeast produce pectinases and bacteria produce acetic and lactic acids



Use of pure pectinases, which yeasts breakdown the citric acid to raise the pH to 4.2 so the lactics can do their job



Which organisms produce the volatiles, oxalic, succinic, phosphoric and malic acids which are precursors to the chocolate flavor



Finally are cannabinoids produced that cause the chocolate cravings.

Pure cultures







D. Miscellaneous Other Processes



1. Mushrooms - OVERHEAD







III. MICROBIOLOGY OF TOPICAL AND OCULAR DRUGS AND COSMETICS





A. Steps in developing a process



B. Fermenters - components, types of flow, why select one over the other



C. Culture preservation - how, why, problems with different methods



D. Types of substances produced by microorganisms, specific examples of different types/classes of compounds and value of Bacillus licheniformis



E. When to harvest, importance of growth stage





REFERENCES



Notes from lecture on control of microbial growth and the references listed there



Farrington, J. K.,, E. L. Martz, S. J. Wells, C. C. Ennis, J. Holder, J. W. Levchuk, K. E. Avis, P. S. Hofman, A. D. Hitchins, and J. M. Madden. 1994. Ability of Laboratory Methods to Predict In-Use Efficacy of Antimicrobial Preservatives in an Experimental Cosmetic. Appl. Environ. Microbiol. 60: 4553-4558.



Section in any general microbiology text on skin



Sharpell, F. and M. Manowitz. 1991. Preservation of Cosmetics. in, S. Block (ed.) Disinfection, Sterilization, and Preservation. Chapter 51, pgs. 887-892. Lea & Febiger. Philadelphia.



Zhou, X. and V. M. King. 1995. An Impedimetric Method for Rapid Screening of Cosmetic Preservatives. J. Indust. Microbiol. 15: 103-107.



Tran, T. T. and A. D. Hitchins. 1994. Microbial Survey of Shared-Use Cosmetic Test Kits Available to the Public. J. Indust. Microbiol. 13: 389-391.



A. Factors Controlling Skin Bacterial Flora



1. pH



2. aw



3. age



4. lipids



Therefore, what are the predominant microbes on the skin and are they the same all over??









B. Nature of the Problem with Cosmetics, Contacts, etc.









C. Nature of the Problem with CAPD (continuous ambulatory peritoneal dialysis) and other home medical devices.























D. Testing



1. Problems



2. Impedimetric - OVERHEADS SHOWING RESULTS





3. US Pharmacopeia Tests and others (Farrington et al. article) - called Challenge Testing: OVERHEADS OF RESULTS





4. Ames Test - measure of the rate of back mutation from a point mutation (reversion)



Salmonella typhimurium - histidine auxotroph; lack DNA repair mechanisms so reversion can only be due to chemical



Test strains: TA 100, TA 99, .....



Activation with Rat Liver preparation for mixed function oxygenases



E. Preservatives



1. Special considerations: toxicity, efficacy, stability, able to withstand repeated challenges



2. parabens







3. quats





4. Kathon CG



















5. co-preservatives







6. BHT/BHA - butylated hydroxytoluene/butylated hydroxyanisole

















7. Dowicil 200









8. Germall II













9. Bronopol











10. DHA