Vinegar production

INDUSTRIAL MICROBIOLOGY

1. Mentor: Ms. Renu NK Jaisinghani Prepared By : Ms. Shivani Shinde Assistant Professor Ms. Priyanka Mahajan Department of Microbiology Mr. Omkar Lobhi Smt. CHM College VINEGAR PRODUCTION

2. VINEGAR PRODUCTION Vinegar fermentation is the oldest fermentation occurring naturally – as unwanted spoilage of Wine . The term Vinegar has originated from Dutch word Vinegar [Sour Wine]. Definition : Vinegar is fermentation derived food product containing not more than 4gm acetic acid per 100 ml of vinegar with specific flavor and aroma at 20ºC.

3. Types of vinegar On the basis of substrate , used for getting alcohol it is named accordingly White vinegar - Grape wine Spirit vinegar - Spirit Alcohol vinegar - alcohol Grain vinegar - grains- wheat, rice, and barley Cider vinegar - apple cider Malt vinegar - barley malt Whey vinegar - whey Fruit vinegar - fruits Sugar vinegar - sugars syrup Glucose vinegar - glucose Rice vinegar - rice starch

4. Vinegar production is 2 steps process- 1) Production of ethanol from sugar using Yeast S. cereviciae var ellipsoid 2) Production of acetic acid (vinegar) from alcohol using acetic acid bacteria Acetobacter aceti Acetobacter curvum Acetobacter orleanense Uses : 1) Used in home for preparation of salads and vegetables. 2) Used as preservatives and flavouring agents e.g. in pickles, vegetables, fish , salads dressing etc.

5. Methods of production: 1) Natural processes or field processes or Let alone processes. 2) French method or slow method or Orleans method. 3) German method or quick method or packed generator ( trickling generator method). 4) Deep fermentation or submerged method or use of acetator . First 3 methods are considered to be surface fermentation as organisms grow as thick mat at the surface.

6. Large amount of oxygen is required as NADH2 released during reaction enters in respiratory chain (ETC) and requires large amount of oxygen for release of ATP, hence if oxygen is less than organisms cannot grow and poor yield is obtained. Biochemistry of Acetic Acid

7. Surface Fermentation : In natural processes, wine with alcohol content 10-13% is taken in open pan and kept for few days at RT. Acetic acid bacteria grow as film (scum) at the surface and carry out fermentation. Not good method - as quality and quantity varies with each batch as no pure culture used or flora varies with every batch. Also less yield is due to limited oxygen available .

8. French fermentation : In this method , fermentation is carried out in big barrels. Initially barrels are filled with pure vinegar and 10-15 liters wine (alcohol 12%). Mixture is incubated at RT, pH =3.5 for 1  week is repeated for 5 weeks and then after 5 weeks 10-15 liters wine is added and 10-15 liters  fermented medium (vinegar) is withdrawal from barrel. Then processes becomes continuous there after. In this method , organisms grown as thick gelatinous mat ( called mother of vinegar ) at the surface. To keep this mat growth at the surface for aerobic fermentation to proceed, wooden raft or Frame is used to support mother of vinegar. Vinegar withdrawn is subjected to storage, aging and clarification. Bottling and pasteurization.

9. Advantage:   Gives fine quality of vinegar. Disadvantage: - Slow hence costly - Long incubation period hence non-acetobacter may grow and produce lactic acid, propionic acid, esters etc. - As use of open barrels there is loss of ethanol by evaporation - Poor yield - Mother of vinegar may get disturbed while addition of wine and this has effect on yield. - It needs lots of attention and checking.

10. German method: 1)In this, aerated medium ( wine with 12% alcohol and pH=3.5)  is inoculated with A. schutzanbachii culture and trickle down through sprinkler over as generator packed with beech wood shaving. 2)Organisms stick to solid support and start growing in the form of zoogleal film . This active zoogleal film of Acetobacter converts alcohol to acetic acid, as it trickles down. 3)Trickling generator has  cooling coils to control temperature and collection chamber at the bottom to collect product. 4)If vinegar collected is not of desirable strength (concentration) then it can be pumped and taken to the top of the generator and recycled till vinegar of desirable strength is obtained.

11. Trickling generator : 1) It consists of air tight tank, 14ft. In diameter and 15 ft. in height. 2) It has flat perforated bottom that divides tank into upper fermenter and lower collection chamber. 3) There is sprayer or disturbing arm fitted near the top of the tank. 4) There is vent at the top and 10 air intakes with air filter situated around the tank near the level of wooden grate. 5) To control temperature, tank is equipped with tubular cooling coils situated at the bottom of the tank in the collection chamber. Thermometers are inserted to control the temperature.

12. Trickling generator : 6) The generator is packed with beech wood shavings upto 1.5ft. from the top of the generator. 7) There are wooden frames inserted in between to support loose packing of wood shavings. 8) Medium ( wine with 12% alcohol + glucose + NH4PO4 + MgSO4 + K- citrate and pantothenic acid  PH-3.5) is sprinkled over packing materials. 9) The vinegar stock or medias mixture is allowed to undergo circulation through beech wood shavings for more and more alcohol to undergo oxidation. This is continued till vinegar of desirable strength is obtained. 10)Conditions for fermentation Temperature - RT pH - 3.5 Time - 10 days

13. Trickling Generator

14. Factors affecting vinegar production : 1) Nature of packing materials 2) Nature of vinegar stock use 3) Rate of flow of ethanolic solution 4) Rate of airflow 5) Temperature Trickling Generator

15. Advantages : 1) higher yield 2) less time 3) being closed generator, no loss of ethanol 4) recycling of vinegar to get vinegar of desirable strength 5) less space required 6) low cost Trickling Generator

16. Trickling Generator Disadvantages : 1) Vinegar eel- Nematodes like Anguillula aceti may enter through dust and insects. They feed on zoogleal film. When large in number , the putrefic a tion of dead nematodes can cause off flavor. 2)Vinegar mites- may also appear in large numbers. 3) Acetobacter xylinum : Contamination by this organism can cause clogging of generator and thereby poor aeration as they produce excess of extracellular  polysaccharide or slime.

17. Deep/submerged fermentation- Main advantages over surface are: High level of aeration necessary for oxidative conversion of alcohol to acetic acid can be provided at low cost Not required compressed air hence economic, low energy input Being closed tank , no loss of ethanol High degree of automation and no manual operation No packing materials hence no clogging Very efficient with quick start up and shut down of fermentation

18. Deep/submerged fermentation- 7) Very high yield 95% 8)Requires less space 9)Oxygen is consumed very fast hence no oxidative browning of raw materials like apple cider or wine mash 10)Low alcohol with less than 6% alcohol can also be used as raw material 11)Vinegar withdrawn at the end of fermentation contains suspended microorganisms which oxides residual alcohol and carry out additional changes in taste, flavor and aroma.

19. Inoculum Development: Lyophilized stock culture of acetic acid Sterile sweet cider with 4% alcohol (R.T/24 hrs on shaker) Step-by-step increases the volume Maintenance of culture is as great problem because these organisms loose efficiency if streaked on solid medium. Also on storage under refrigeration organisms do not survive for long. Hence , organisms are maintained on, Sterile tomato juice agar slant in screw capped tubes with small amount of sterile broth. Lyophilized stock. Maintained in liquor fermentation medium (small fermenter)

20. Fermentation Medium: Alcohol - 13% Vinegar - to adjust pH to 3-3.5 Small amount of glucose, NH 4 PO 4 ,MgSO 4 , K-citrate, pantothenic acid Alcohol concentration should be between 10-13%.At higher concentration acetic acid bacteria may get inhibited and at lower concentration poor yield because of complete oxidation of alcohol to CO 2 and water. Also ,loss of aroma and flavor because of oxidation of ester .

21. Fermentation Conditions: Temperature: R.T. is optimum. At low temperature: process is slow and time required is more. At high temperature : loss of ethanol by evaporation and decrease in efficiency of organisms. No development of flavor and Aromas Oxidative process of vinegar production is exothermic hence lots of heat generated and temperature increases. Efficient cooling system is required. 2) pH: optimum 3-3.5 It favors growth of acetic acid bacteria and prevents growth of contaminants.

22. 3) Oxygen supply: From biochemistry of production of vinegar it is clear large amount of oxygen is required for generation of ATP and for oxidative process. Acetator used has special aeration device that make use of fresh air and bring about efficient aeration at low cost and energy input. Also, vigorous and uniform aeration is achieved. 4) Time: Two days and operated in semi continuous fashion. 5) Yield: 15 gm % After fermentation, vinegar withdrawn from fermenter is subjected to storage, aging, clarification, bottling and pasteurization.

23. Post harvest steps 1.Storage: Vinegar withdrawn is stored in barrel upto the rim Barrels are sealed to avoid contact with atmospheric oxygen During storage further oxidation by organisms is carried out of vinegar to ester, which gives flavor and aroma but care is taken to avoid complete oxidation of vinegar to CO 2 and water. This results into decrease in strength of vinegar.

24. 2 .Aging: Process that improves flavour and clarity of vinegar. During aging also esters are formed that add to flavour and aroma and also reduce harsh flavour and odour. 3. Clarification: All suspended particles and microorganisms are removed if not they may give turbidity or haziness to final product. This can be done by- Filtration - RVF By adding fining agents in proper proportion(Bentonite, Spanish clay= 1to 5 lb/100 gals of vinegar)mixture is allowed to stand till as clear vinegar appears at top. Post harvest steps

25. 4. Bottling and pasteurization: Bottles are completely filled with vinegar and tightly capped or corked to prevent entry of air. Bottles are then pasteurized at 60-66˚C for 30min. 5. Units used to express concentration of acetic acid in vinegar Vinegar grain gallon : 1 gal of 100 grain (10% acetic acid) Vinegar grain strength : 1 grain vinegar is 0.1 gm of CH 3 COOH in 100 ml of vinegar at 20˚C i.e. (10mg/ml) or ( 0.1 gm%) Post harvest steps

26. Fringe Acetator:(Design and working) It was 1 st described by Hromatka Acetator means excellent performance by the fermenter It gives high yield 15gm% at low cost It is cylindrical stainless steel fermenter i.e. 4m. in diameter and 9 to 13m in height. It is operated in semi-continuous fashion.

27. IMPORTANT PARTS OF ACETATOR Acetator used device for aeration consists of Hollow body turbine surrounded by a stator. Hollow body is connected to fast rotating (1400-1700 rpm)ceramic disc in clockwise direction. It has 6 openings for escape of air. Arranged radially and open against direction of rotation. Ceramic disc has upper and lower ring and upper and lower stator for support. This stator rings are connected with vertical baffles. Baffles direct air bubbles in upward directions. The other end of Hollow central pipe is connected to air suction. Because of proper size and speed of rotor ; sufficient air get aspirated and dispersed outward radially in form of tiny bubbles. It works by self-aspiration and doesn’t required compressed air .

28. Acetator-

29. Fringe defoamer- Due to vigorous aeration lots of foam is formed. Chemical Antifoam agents are not used as final product is food product, mechanical defoamer is used. Defoamer mounted at the top of acetator. It consists of spiral housing in which rotor with wings is fitted. This rotor rotates at 1000 rpm . As foam enters in spiral housing due to centrifugal force of rotor, foam is thrown at periphery and break gases released then leave the fermenter through gas vent and liquid is drawn back to fermenter.

30. Fringe’s alkograph It is automatic instrument for measuring the percentage of alcohol in fermentation medium so as to control complete process. It is made up of 2 analyzers vessels. Amount of fermentation medium flow continuously through it .When it enters in the 1 st vessel; it boils till all ethanol evaporates. This boiling point is recorded (A) it is less as medium is mixture of water and alcohol.

31. Then liquid flows to 2 nd vessel and boils again. Its boiling point recorded again (B) this is always higher as it mainly water in its. The difference between two boiling points (B-A) gives % alcohol present in the medium and is recorded automatically. As fermentation proceeds, % alcohol decreases. The other parameters are also controlled automatically on the same basis. i.e. When alcohol concentration drops down to 0.2- 0.3 % alkograph gives signal and control the alcohol percentage . Fringe’s alkograph

32. Yeoman’s Cavitator

33. Yeoman’s Cavitator Discovered by Yeoman‘s company. It operates in continuous fashion . Aerator is made up of simple hollow body turbine with arms and paddle, this driven from top using magnetic coupling . Hollow shaft that helps in rotation of aerator. Cylindrical body around the shaft suck liquid, foam and air from headspace and draws this emulsion to bottom of the tube, thus cavity is formed. Air trapped in cavity is dispersed through medium.

34. Reference Casida L. E., "Industrial Microbiology” (2009) Reprint, New Age International (P) Ltd, Publishers, New Delhi. Crueger W. and Crueger A. (2000) "Biotechnology -"A Textbook of Industrial Microbiology", 2nd edition, Panima Publishing Corporation, New Delhi. Prescott and Dunn's ‘’Industrial Microbiology’’(1982) 4th edition, McMillan Publishers

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