The Development of Inocula for Industrial Fermentations and Wine Spoilage Organisms

Lecture 3

What criteria should fermentation inoculum satisfy?

It must be in a healthy and active state Available in sufficiently large volumes to provide an inoculum of optimum size Suitable morphological form Free of contamination

Retain its product-forming capabilities

What is difference between Inoculum and Production medium?

Inoculum grows in culture medium Design of production medium is determined not only by nutritional requirements of organism but also by requirements for maximum product formation Both media can be of different composition

Examples of inoculum and production media

How much quantity of inoculum is used?

Volume : Normally 3 10% is used Stock culture Inoculum built up in number of stages Risk of contamination and strain degeneration

Criteria for transfer of inoculum

Physiological condition of inoculum major effect on performance of fermentation Indicators of inoculum quality include dissolved oxygen, pH and Co2

Yeast, Bacteria and Fungi common inocula

Preservation of Industrial Microorganisms

Stored eliminate genetic change, protect against contamination and retain viability Storage at reduced temperature agar slopes (5) or freezer (-20) SC 6 months

- Storage under liquid nitrogen resuspension in 10% glycerol , -150 to -196C

Preservation of Industrial Microorganisms

Storage in a dehydrated form - Dried soil cultures fungi and actinomycetes - Moist sterile soil inoculate for several days for some growth dry at room temperatures for 2 weeks refrigerate samples - Lyophilization or freeze drying Bacteria - Freezing culture followed by drying under vacuum resulting in sublimation of water.

How do Yeast multiply?

Asexual Fission or Budding - Takes place within a single parent (haploid or diploid)

- Splits nucleus forms daughter cells

How do Yeast multiply?

- Sexual haploid cellsShmooing form Diploid cells - Unfavorable conditions haploid cells die - Diploid cells undergo meiosis - into 4 daughter haploid spores

Development of yeast inocula

Wort Batch of fermentation Crop Harvested yeast from previous fermentation Pitch Inoculate

Development of yeast inocula

Middle skimmings required Yeast cells float to the surface most flocculate - Bottom cells least flocculate - Skimmings treated reducing pH of slurry to 2.5 to 3, washing with water or ammonium persulphate and treatment with antibiotics like Polymixin, Penicillin and Neomycin

How do Bacteria multiply?

Asexual reproduction Bacteria splits into two cells Binary Fission Sexual reproduction Joining of two parent cells and exchange of genetic material - Conjugation, Transformation and Transduction

Development of bacterial inocula

Produce active inoculum gives short lag phase Should be transferred in logarithmic phase of growth metabolically active Small amount of inocula prevent contamination and avoid an abnormal long lag phase

How do Fungi multiply?

Asexual reproduction forms spores Sexual reproduction Two parent cells unite to form zygote

Development of mycelial inocula

More difficult than yeast and bacteria Industrially important fungi and streptomycetes capable of asexual sporulation use spore as inocula

Development of mycelial inocula

Three basic techniques produce high concentration of spores for use as inoculum - Sporulation on solidified media - Sporulation on solid media

- Sporulation in submerged culture

Development of mycelial inocula

Sporulating on solidified media:

Development of mycelial inocula

Sporulation on solid media - Many filamentous organisms will sporulate on surface of cereal grains - Substrates barley, hard wheat bran, ground maize and rice - Affected by amount of water added to cereal before sterilization and relative humidity

Development of mycelial inocula

Sporulation in submerged culture: More convenient than others easy to operate aseptically and done on large scale Suitable medium

What is Wine fault/defect?

Unpleasant characteristic of wine Poor winemaking or storage conditions leading to wine spoilage. Compounds positive characters to wine - Varying conc. replace or obscure the flavors and aromas

What factors lead to wine spoilage?

Oxidation Sulfur compounds Environmental conditions Microbiological agents

How does Oxidation lead to Wine Spoilage?

Storage position of bottles - Storing bottles vertically creates a heterogeneous environment Horizontal storage - the wine competes with the bacteria for oxygen Formation of acetic acid, lactic acid and ethyl acetate

How do Sulfur compounds lead to Wine Spoilage?

H2S is a metabolic by-product of yeastfermentation in nitrogen limited environments.

Mercaptans by reaction of hydrogen sulfide + ethanol or sulfur containing aa Diemthyl sulfide is formed from breakdown of sulfur containing aa.

How do environmental factors lead to Wine Spoilage?

Heat - overcook wine Light strike excessive exposure to UV 325450nm wet wool or cardboard flavour and aroma

Ladybird beetles release unpleasant volatile compounds into wine.

What are the three types of yeast wine spoilage organisms?

Type Type A Occurs in Affects on wine Sweet white table Loses sugar, sparkles, wines yeasty taste

Type B

Dry white table wines

Type C

Yeast sedimentation, no gas absence of sugar Sweet and Dry Flaky or granular white table wines yeast, no gas, no turbidity, no yeasty taste

What are the various Yeast wine spoilage organisms?

Yeast Brettanomyces bruxellensis Release Changes in wine Off-flavors Volatile acids, esters and phenols like 4ethylphenol and 4ethylguaiacol Zygosaccharomyces Resistant to high sugar Pichia and Volatile acids Hanseniopsora acetic acid and ethyl acetate

Turbidity and CO2 gas Films formed in barrels and tanks

What are the various Bacteria wine spoilage organisms?

Pediococcus Polysaccharides Increase in like biogenic viscosity due to amines - histamine slimy or fat taste graisse

Lactobacillus (malolactic bacteria)

Acetic acid

Mousy flavors Mousiness

Gluconobacter and Acetic acid in Volatile acidity Acetobacter (acetic presence of SO2 & acid bac.) O2 Oenococcus oeni Diacetyl compounds Intense buttery or butterscotch

What are the various Molds wine spoilage organisms? Molds like Steptomyces, Botrytis cinerea and Penicillin expansum (on grapes) provide earthy, musty, beetroot, even turnip flavour and aroma to wine.

Which enological operations sources of contamination?

Airborne contamination dust and flying insects - Pass through cellar treatments bottling - Filter materials, bottles, corks, hose-lines, tank fittings, filler tubes and other exposed points - The yeast can fall directly into mouths of bottles on the bottling line between filler and corking or capping machines

Which enological operations sources of contamination?

Spoilage yeasts exist in wine before bottling Natural flora present on grapes

What are the different ways of microbial analysis?

PCR, Sequencing etc Biochemical tests malic acid, glucose and fructose concentrations Volatile phenols gas chromatography Chemical analysis pH, acidity, alcohol content, SO2 and total polyphenol index

What are the different winery practices needing checking?

Nature and amount of spoilage (sediment in bottle, microscopic appearance of yeasts, amount of spoilage, temperature range, time for development, sulfur dioxide, pressure in bottle) Sanitary condition of winery and equipment: (winery surroundings, fermenting cellar, storage cellars, bottling room and equipment) Cellar practices preparation for bottling and the bottling process

Control methods to stop growth of spoilage organisms

Sulfur dioxide - good method of control for many undesirable yeasts. Prebottling filteration - coating the cloth or pads on a filter press with a suitable amount of filter aid (diatomaceous earth) of proper grade Electronic pasteurization population of one million cells per cc can be killed by exposure to an electronically induced temperature of 130 F for less than 4 seconds.

Primary articles referred to:

1. Sources of Spoilage Yeast in Bottled Wines John G. B. Castor Am. J. Enol. Vitic., Jul 1951; 2: 31 - 42. 2. Survival of Wine Microorganisms in the Bottle during Storage Vincent Renouf, Marie-Claire Perello, Gilles de Revel, and Aline Lonvaud-Funel Am. J. Enol. Vitic., Sep 2007; 58: 379 - 386. 3. Yeast Spoilage of Wines: A Review of the Problem Yohn G. B. Castor Am. J. Enol. Vitic., Jul 1952; 3: 139 - 159. 4. Practical Implications of Malolactic Fermentation: A Review C. R. Davis, D. Wibowo, R. Eschenbruch, T. H. Lee, and G. H. Fleet Am. J. Enol. Vitic., Dec 1985; 36: 290 - 301. 5. Clouding of Wines Due to Micro-Organisms Myron C. Nightingale Am. J. Enol. Vitic., Jul 1953; 4: 85 - 89. 6. Killer Yeasts in the Wine Industry: A Review H. J. J. Van Vuuren and C. J. Jacobs Am. J. Enol. Vitic., Jun 1992; 43: 119 - 128. 7. Airborne Microbial Contamination in A Winery Bottling Room David M. Donnelly Am. J. Enol. Vitic., Sep 1977; 28: 176 - 181.

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