LACTIC STARTER CULTURES AND BACTERIOPHAGES

Transcription

1 LACTIC STARTER CULTURES AND BACTERIOPHAGES INTRODUCTION Selected strain of food-grade microorganisms of known and stable metabolic activities and other characteristics that is used to produce fermented foods of desirable appearance, body, texture, and flavor. Some are also used to produce food additives (organic acids, bacteriocins) probiotics drug delivery. Give better product than those produced through natural fermentation of the raw materials. During successive transfer of mother starter to fresh medium often face bacteriophage infection that brings product failure if careful procedures are not taken. Mainly used in dairy industry (yogurt, fermented milks, cheese) LAB STARTER FUNCTION Fermentation of sugars organic acids ph decrease clotting, reduction and prevention of adventitious microflora Protein hydrolysis texture change, taste enhance Synthesis of flavor Synthesis of texturing agents Production of inhibitory components (i.e., bacteriocin, etc) Consistency Prevent product failure Industrial scale production HISTORY Initially back slopping and natural fermentation Still used in domestic manufacture of fermented milk.

2 Back slopping Before 1950 s Stock culture mother culture bulk culture (1-2 % of volume of milk) Difficult to produce of consistent quality Small business Single strains of known Starter failure due to bacteriophage After 1950 s Combating against phage Rotation of strains Multiple strains Defined media to produce bulk starter to reduce phage attack Consistent quality After 1960 s Introduction of frozen concentrate culture that have high cell number ( cells/ml). Could be directly inoculated into milk (direct vat set, DVS) unnecessary to produce bulk starter. Air transported in dry ice. Phage inhibitory media (PIM): high concentration of phosphate (PO4 - ) to chelate calcium (Ca 2+ ) in milk unable phage to adsorb on bacterial cell.

3 After 1970 s Demand increased Different types of starters Cheese, yogurt, fermented sausage, some ethnic fermented foods Freeze-dried concentrated cultures Eliminate use of dry ice Prevent accidental thawing But some cells do not survive well in freeze-dried state. Recently Custom designed starter cultures Understanding genetic background of some important desirable traits, phage inhibition TYPES OF STARTER CULTURES TRADITIONAL STARTER LIQUID STARTER Scale-up: Liquid stock Mother Intermediate Bulk (increase volume by successive subcultures) Expensive Laborious Needs skillful personnel Easily contaminated by bacteriophages Still practiced FREEZE-DRIED CULTURE The same as liquid culture for preparation Used when small amount of starter is needed Excellent preservation (@ - 25 C for several months) Mixed stain should be separately freeze-dried to maintain balance between strains

4 CONCENTRATED CULTURES (DIRECT VAT SET, DIRECT VAT INOCULATION) CONVENTIONALLY In controlled fermentation, starter is ca cells/ml. In conventional bulk starter with ca cells/ml needs to be added at 1% (v/v) to the milk For 100,000 gal of milk, > 1000 gal (1% of 100,000 gal) of bulk starter is needed daily. Too much volume to handle!! This large volume is produced from mother culture through several intermediate subcultures (more handling at processing facilities) vulnerable to phage infection since phages are more abundant in processing environment (Fig. 13-1). SOLUTION Instead, more handling is done by culture producers under controlled environmental conditions, minimize phage problems. Let the pros take care of the job!! FROZEN CONCENTRATE AND FREEZE-DRIED CONCENTRATE Skip mother, intermediate, and bulk Possible to direct inoculation to production process Easy to use Good starter activity Less labor Bacteriophage problem is limited Significant savings in labor, material costs Drawbacks: Require low temperature during shipping and storage (dry ice in Styrofoam box)

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6 Concentrate the cell by centrifugation up to cells/ml add cryoprotectant (DMSO, glycerol) and freeze store in dry ice (- 78 C) or liquid nitrogen (-196 C) distribute in Styrofoam -20 C or below thaw in warm (45 C) de-chlorinated boiled

7 water before use. Only 360 ml of frozen concentrate culture in DVS system can be added to 5,000 gal milk to get desired cells/ml. Or the concentrated cell can be freeze dried plastic bag under vacuum distribute to factory store < 5 C or in refrigerator (use it within expiration date, usually for 3-12 mo) use. Dairy industry Several additional steps: Phosphate buffer Minimize acid damage Protect bacteriophage BULK CULTURE

8 Low lactose level Limit acid production Reduce acid injury Food grade ingredients STARTER CULTURE PROBLEMS STRAIN ANTAGONISM In mixed culture (two or more strains) Dominance strain due to different growth environment or production of inhibitory metabolites (e.g., bacteriocins, acids, peroxide). Use compatible strains. LOSS OF DESIRED TRAITS Plasmid-linked traits (lac +, cit +, muc +, bac +, R/M, suc + ) are usually lost during storage, subculturing, and under some nonselective growth conditions. Physical (e.g., freezing and thawing) and chemical stress also result in loss of desirable traits. Genetic studies (i.e., integration of plasmid mediated genes on chromosomal DNA) are being conducted to understand the mechanism. CELL DEATH AND INJURY The effectiveness of freeze-dried concentrated or frozen concentrate starter depends on two important factors: Culture has to have large number of viable cells Cells should have a short lag phase so that they can multiply quickly in food. To minimize cell damage (cryoinjury) to cell: Addition of cryoprotectant Rapid freezing at very low temperature To minimize cell viability loss, avoid: Repeated freezing and thawing Thawing long before use

9 Mixing starter with curing salts, spice for a long time (sausage fermentation) Long -20 C or higher temperature. INHIBITORS IN RAW MATERIALS Antibiotics and sanitizers in milk Phosphate or nitrite in sausage fermenting factories INDUSTRIAL SCALE PRODUCTION OF A. FERMENTATION STARTER CULTURE GROWTH MEDIUM Should be cheap but contain enough nutrients for the growth Should contain some milk solids to ensure the synthesis of necessary enzymes for starter to perform well in milk. Cheese whey and whey permeate w/ supplements Cheap medium (waste product) Limitation for some nutrients for maximum growth Need partial hydrolysis by proteolytic enzyme to improve growing Precipitate after pasteurization clarification step should be followed Not considered adequate for maximum growth Skim milk supplemented w/ sodium citrate (solubilize milk proteins helps harvesting of cells) Same composition of cultured milk products Good choice medium Contains milk solids Maintaining balance among strains in multiple strain starter Yeast extract Peptone Growth factors (if needed) Tween 80 (polysorbate 80, polyoxyethylene monooleate):

10 nonionic surfactant and emulsifier Oleic acids (C19, unsaturated FA, membrane fluidity) GROWTH CONDITIONS Optimum growth temperature Optimum ph Neutralizer: ammonium hydroxide Cooling Harvesting time: end of log phase Oxygen toxicity (due to constant agitation to maintain constant ph): Reducing agents Catalase CO2 sparging PH CONTROL Cells held at ph 5 or below for even modest period of time will lose viability and behave sluggish when inoculated into fermentation tank. Preventing acid injury during the production and propagation of starter culture is essential. Also increase cell density. Two approaches External ph control: monitoring ph and addition of neutralizing agents (NaOH or KOH) maintain ph Internal ph control: addition of buffering salts (NaCO3, Mg3PO4) slowly become soluble in medium maintain ph > 5

11 HARVESTING CELLS (CONCENTRATION) Centrifugal separation or membrane process Ultrafiltration Microfiltration w/ ceramic filter HANDLING AND STORAGE Cryoprotectant Lactose Sucrose DMSO (inapplicable to food) Glycerol Low temperature (-196 C for LN2, deep freezing, etc) The lower the temperature, the longer the shelf life Monosodium glutamate (MSG) provide some protection to cultures during freeze-drying ENCLOSED STARTER PROCESSOR This specially designed process vessel provides a high degree of protection for the production of bulk starter and minimizing chances for bacterial and phage contamination. It is a completely sealed unit and includes the following: An air-tight, insulated cover with stainless steel hinge and cam latch, an O-ring gasket snaps on the cover. A removable inoculating port with live steam ring and sealed cover for protective inoculating technique.

12 Easily attached, high-efficiency filter for controlled entrance of air and protection from airborne contamination. Sealed agitator port with two-piece rotary agitator seal. Complete seal can be removed quickly and easily without removing agitator shaft. FUTURE TRENDS SEVERAL STRAINS (MIXED STRAINS) Relationship Symbiotic and no inhibitory phenomena between strains (compatible) GENETICS Good flavor + acid + bacteriocin producer enhance safety Uncooked fermented foods (fermented sausages, kimchi, sauerkraut) Identification of corresponding genetic determinant of certain desirable traits Stable gene transfer methods Customized starter Isogenic gene transfer (within the same species via conjugation) no GMO. GRAS Full genomic sequence of lactic starters BACTERIOPHAGES OF LAB

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14 Major commercial problems in dairy fields: Mostly cheese fermentation (open system)

15 Phage survive pasteurization of milk Once contaminated in dairy environment, easily spread by air, whey residue Product failures (symptoms): Unacceptable low production of lactic acid and flavor Reduced proteolysis Slow vat Dead vat Severe economic losses (cheese industry is more serious, open fermentation system) 20 temperate and virulent bacteriophages have been found and their genomes have been completely sequenced. Caudovirales: tailed phages Most of them carry kb genome Cells carrying prophages are resistant to attack by temperate phage and thus will be dominant in the population G + C content of the phage genome is similar to the G + C content of the bacterial host chromosome reflecting intimate phage/host relationship

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17 PHAGE LIFE CYCLE

18 COMMON STEPS PHAGE ADSORPTION (REVERSIBLE) Phage specific receptors on host: Carbohydrate components in cell wall (Rhamnose is essential) S-layer protein Need Ca 2+ or Mg 2+ PHAGE PENETRATION (IRREVERSIBLE) Mediated by phage infection protein (PIP) located in cell membrane Transmembrane protein required for phage DNA injection CIRCULARIZATION

19 HOSTS DEFENSE MECHANISM AGAINST PHAGE 4 NATURALLY OCCURRING MECHANISMS ADSORPTION INHIBITION Masking/Shielding Production of exopolysaccharide (EPS) or capsular polysaccharide (CPS) BLOCKING OF PHAGE DNA PENETRATION Some mutant lack of phage infection protein (PIP) in cell membrane Still adsorb phage, but defective in penetration through membrane RESTRICTION/MODIFICATION (R/M) SYSTEM Restriction endonuclease Methylation Plasmid linked However, DNA progeny that has escaped restriction will reinfect at higher efficiency

20 Efficiency of R/M depends on number of recognition sites in phage DNA. ABORTIVE INFECTION (ABI) SYSTEMS Last crucial step for host to combat phage attack Premature host cell death prevent early or late in lytic cycle of phage development Absence of plaque or severe reduction in plaque size (titer) and burst size Plasmid encoded Unusually low G+C content of their genes Supplemented by R/M system incidence of cell death due to Abi systems will be minimized Some mutant phages overcome this system ARTIFICIAL PHAGE RESISTANCE MECHANISMS (MOLECULAR) ANTISENSE RNA STRATEGIES: A second sequence of RNA complementary to the first strand (mirror image of mrna). The formation of double stranded RNA can inhibit gene expression in many different organisms including plants, flies, worms and fungi. 5 C A U G 3 mrna 3 G U A C 5 Antisense RNA Cloning of antisense RNA behind promoter will bind target sense mrna Prevent translation of phage proteins by either destabilizing and making more susceptible for degradation by RNase or by inhibiting loading of ribosome

21 Figure 1 Formation of antisense RNA blocks translation. PER Phage Encode Resistance Utilization of phage origin of replication (ori, recognition site for phage DNA replication)) Cloning of phage ori in multi-copy plasmid vector (multiple decoy copies) transform host cloned per competes in trans with normal phage replication the greater the expression of the plasmid, the more phage resistance Increase in insensitivity of host to phage is enhanced Highly effective but specific for distinct type of phages CLONING OF PHAGE REPRESSOR GENE FROM TEMPERATE PHAGE Protection against superinfection of the same temperate phage But not against virulent phages CONJUGAL TRANSFER OF PLASMID-LINKED PHAGE RESISTANCE SYSTEMS Not regarded as GMO

22 CLONING OF ABI AND R/M USING PHYSIOLOGICAL SELECTION MARKER Suicide trap We need good selection marker Bacteriocin (Nisin) resistance marker (nis r ) is linked with phage insensitivity determinant

23 Constructing plasmid containing Abi and R/M Promoter of lactococcal phage:lactococcal R/M gene Phage infection induction of R/M lyse host DNA single cell death (altruistic death), programmed cell death (only infected, not the whole population) but phage propagation is blocked sparing remaining cell from more severe phage infection PHAGE CONTROL (NON-MOLECULAR) DEFINED STRAIN Check the presence of prophage before use by mitomycin C induction Professional starter producer PROPER SANITATION CIP chlorine, hypochlorite Sterilization of whey (major bacteriophage reservoir) Isolation of culture handling area from rest of processing facility Maintaining positive pressure for culture handling area/ air filtration phages are frequently transmitted via air or airborne droplets Do not let whey contaminate starter culture Phage insensitive media (PIM): Chelating Ca 2+ by adding phosphate or citrate Rotation of starters Mixed strains Slow-acid producing strain (prt - ): generally less susceptible to phage attack than fast-acid-producing strains (prt + ) ph-controlled system Phage resistant starter strains: Grow the starter with lytic phage select cells (spontaneous phage-resistant mutants) that were not killed by phage apply to processing (Heap-Lawrence test) Constant scrutinized phage monitoring Detection of phage DNA in whey by dot blot technique PCR ELISA

24 Mitomycin C induction of temperate phage before use

25 Plaque Validation of phage resistant strains at pilot and commercial scale. In the trial, the phage resistant strain performed well under manufacturing conditions and resulting 6-month-old cheeses received flavor scores equal to, or better than, cheeses made with

26 the parent strain. This strain was subsequently validated in a commercial cheese plant where it yielded a product of exceptional quality. In addition, subsequently, the phage resistant derivative of the strain DPC4932 (DPC5020) was validated in two commercial cheese plants. Phage therapy ( 숙제 )