Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/54—Acetic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid

Definitions

• the present invention relates to a method for simultaneous preservation and pre- treatment of plant juice and other aqueous agricultural residues to result in a stable fermentation medium which can be used for the production of e.g. organic acids, amino acids, probiotics and enzymes.
• the present invention involves an environmentally friendly and economically feasible "bio- refinery” which is a further development of existing industries where the production is based on agricultural crops and where waste materials and residues are used for produc- tion of new products in integrated processes. Examples of such industries are the green crop drying industries, potato starch industries and sugar factories.
• typical crops for such purposes are grass, clover, alfalfa, sugar beet, potatoes, Jerusalem artichoke (Helianthus tuberosus), corn and grain crops.
• the green crops i.e. grass, clover, alfalfa and stems from Jerusalem artichoke, can be har- vested in several cuts, cut into small pieces and transported to the bio-refinery, where they first will be subjected to a wet separation to result in a press cake and a juice ("green juice").
• the press cake can be utilised in the green crop drying plant for production of green pellets for animal feed, whereas the green juice is a residue that in principle can be used as fertiliser.
• the season for such a production in the temperate zone is normally from about May to November.
• Potatoes are typically harvested in the temperate zone from about September to November and can thus be utilised in the bio-refinery from about September to February.
• the potatoes are washed, ground and separated into starch, pulp and juice. If juice to which inorganic acids have been added is heated, the proteins therein will precipitate and can be separated from the juice by a decanter centrifuge.
• the deproteinised potato juice is the residue from this production, and it can be used as a fertiliser as it is or can be concentrated by evaporation and used in concentrated form as animal feed or as fertiliser.
• Green juice (unheated) and brown juice (heated) from the green crop drying industry and potato juice from potato starch factories are in most countries used as a fertiliser for crop plants, since these plant juices have a valuable content of nutrients such as potassium and nitrogen.
• nutrients such as potassium and nitrogen.
• Potato juice normally has a dry matter content of about 2-4.5%, depending on the separation technology used, whereas juice from green crops such as grass, clover grass and alfalfa normally has a dry matter content of about 3-10%, depending on the crop, season, weather conditions and wet separation technologies. From a theoretical point of view they are all well suited as fermentation media, as they contain all the necessary growth factors such as amino acids, vitamins and minerals, but in practice it has not been possible to utilise such plant juice in an optimal manner due to environmental and/or economic reasons.
• the problem addressed by the present invention is thus how to successfully utilise these waste products in an environmentally and economically acceptable manner, in particular how to turn waste products into useful products without simultaneously creating new waste products
• WO 92/19716 discloses a method for obtaining a nutrient medium from plant juice, by subjecting the plant juice to a heat treatment, cooling the heat-treated juice, adjusting the pH to 7 5-8 5, and subjecting the juice to hydrolysis using proteolytic enzymes and under the addition of ammonia
• the method of WO 92/19716 has the disadvantage, however, of being expensive and requiring a large amount of energy
• Japanese unexamined patent publication (Kokai) No 59-179036 describes a method for producing protein feed by pressing green plants, inoculating the resulting juice with a lactic acid bacteria such as Lactobacillus and subjecting the juice to anaerobic fermentation under a nitrogen atmosphere
• the fermented juice is then separated by centrifugation into a fraction containing coagulated protein and lactic acid bacteria cells and a residual solution (brown juice)
• the brown juice is inoculated with a yeast such as Candida and aerobi- cally cultured followed by e g centrifugation to result in a waste solution and a yeast fraction, the latter being mixed with the coagulated proteins recovered earlier and dried to form a protein feed
• the present invention provides a simple, inexpensive and effective method for treating plant juice to result in a stable product that can be used as a complete, basic, universal fermentation medium for the production of useful fermentation products
• the method of the invention has the advantage that it results in a variety of useful products without creating new waste products in the process
• the fermentation products that may be produced according to the invention include, but are not limited to, 1) organic acids such as lactic acid and acetic acid, 2) ammo acids such as L-lysme and L-threonine, 3) special feed additives containing both lactic acid and ammo acids, e g L-lysme lactate, and 4) feed enzymes and other fermentation products, including other enzymes as well as proteins and peptides
• the fermentation medium is also suitable for cultivation of cultures of microorganisms such as yeasts, moulds and other fungi as well as bacteria, for example for use as a probiotic in animal feed
• the invention may be used for producing functional feed or a functional feed additive
• the term "functional feed" refer
• the fermentation products produced according to the invention may be used for various industrial purposes
• industrial uses include de-icing compounds (salts of organic acids, e g calcium magnesium acetate) as well as chemicals for use in industrial production processes (e g lactic acid for the production of lactic acid-based polymers, etc )
• chemicals for use in industrial production processes e g lactic acid for the production of lactic acid-based polymers, etc
• Other examples include lysine lactate for use in the cosmetic industry and ethyl lactate for use as an environmentally friendly "green" organic solvent
• the invention comprises an integrated process that can be based on different residues and waste products, treated together or separately, and all derived from the agro-industry
• the residues or waste products are pre-treated with the aim of preserving the product before use as a fermentation medium and to release fermentable compounds and growth factors, so as to end up with a stable and highly useful fermentation medium
• the invention is based on the surprising fact that it is possible, by means of a simple and inexpensive method, to utilise a very large proportion of the existing waste products and residues from agro-industrial productions for the production of fermentation media
• An important advantage of the method of the invention is that it results in a complete fermentation medium which need only be supplemented with specific growth factors (e g a carbohydrate source for the production of lactic acid or acetic acid, and ammonia for the pro- duction of ammo acids) in order to form an ideal fermentation medium in which essentially all organic as well as inorganic compounds are utilised for production of cell mass and fermentation products.
• specific growth factors e.g a carbohydrate source for the production of lactic acid or acetic acid, and ammonia for the pro- duction of ammo acids
• addition of e.g. amino acids, vitamins, minerals, etc. is normally unnecessary.
• the end product includes the microbial biomass and the organic compounds present in the fermentation broth, all of which can be used e.g. as a feed additive.
• the invention involves pre-treatment of plant juice to provide an acidified and deproteinised plant juice which is subsequently used as a fermentation medium.
• the juice is acidified and deproteinised by means of lactic acid fermentation, followed typically by sedimentation, or heating and centrifugation, and removal of sedimented protein.
• Another possibility is to obtain acidification by adding an acid to the plant juice, followed typically by heating, centrifugation and removal of protein.
• the juice can optionally be concentrated by e.g. evaporation. The result is a useful and storage-stable product that can be stored as is or in concentrated form, after which it can be sterilised if necessary and used for further fermentation.
• the invention thus makes it possible to convert otherwise worthless and environmental costly waste products and low price residues to useful fermentation media with a significant added value.
• These fermentation media can be used in a wide variety of fermentation processes, but they are especially interesting in cases where the price of the fermentation medium accounts for a substantial proportion of the production cost of the product. This is the case for products such as organic acids used for production of polymers, for example polylactate, and amino acids such as L-lysine and L-threonine, as well as enzymes and probiotics used in animal feed.
• the invention relates to a method for treating an organic waste product comprising plant juice, the method comprising a) providing an acidified and deproteinised plant juice having a pH of less than about 4.5, and b) using the acidified and deproteinised plant juice as a fermentation medium in at least one fermentation step so as to result in a useful fermentation product without creating a new waste product that must be subjected to further treatment
• Additional aspects of the invention relate to fermentation products produced according to the method of the invention and to an animal feed or feed additive comprising such a fermentation product
• plant juice is intended to encompass any juice obtained from any suitable plant or part thereof
• the invention is directed to methods based on the use of fresh or untreated plant juice, where "fresh” or “untreated” plant juice refers to juice that has not been subjected to a sterilisation treatment
• fresh juice includes both "green juice” obtained by pressing fresh plant biomass as well as “brown juice” obtained by heat-treatment of plant biomass
• the plant juice used according to the invention may contain not only dissolved or suspended components, but also may contain additional plant residue The presence and amount of such plant residue, as well as the form of any such plant residue, will of course depend on the particular crop and the method by which it is harvested, as well as possible post-harvest treatments such as cutting, chopping, grinding, etc
• the plant juice will contain liquid as its major constituent, which means that the methods described herein do not encompass e g known fermentation methods for producing silage
• the plant juice used as the raw material for purposes of the invention will normally be a waste juice that is not suitable per se for consumption by humans or animals
• the invention does not encompass known processes for e g malolactic fermentation of fruit juice or fermentation of vegetables for the production of "Sauerkraut" or similar products
• the product resulting from the fermentation medium prepared according to the invention will typically be a non-food product (i e a product not designed for human consumption), for example an animal feed additive or a product for industrial use
• the plant juice to be treated according to the invention preferably has a carbohydrate concentration of at least about 2% by weight (w/v) However, it is also possible to use plant juice having a carbohydrate concentration of less than about 2%, in which case the plant juice will normally be supplemented with a carbohydrate source to result in the desired carbohydrate concentration of at least about 2%.
• fresh plant biomass for example grass, clover or alfalfa
• biomass that has been stored for a short time in the field e.g. a few days, such as one to two days
• the juice obtained from the pressing is preferably acidified by lactic acid bacteria.
• the term "acidified plant juice” refers to plant juice which has been treated so as to have a pH of less than about 4.5.
• the acidified juice will typically have a pH of about 4.0 or less, e.g. from about 3.5 to about 4.0.
• a preferred method for obtaining acidified plant juice is by means of lactic acid fermentation, since this method is easy, inexpensive and efficient.
• acidification of the plant juice may be performed using a conventional method involving addition of acid to obtain a desired pH under about 4.5, typically followed by heating to a temperature in the range of about 80-120°C, e.g. about 90-110°C, and removal of protein using e.g. centrifugation.
• the acid added in this case to obtain acidification may be any suitable inorganic or organic acid, for example hydrochloric acid, sulphuric acid, phosphoric acid, formic acid, lactic acid, etc.
• deproteinised plant juice refers to juice from which all or almost all of the protein has been either removed or partially hydrolysed to smaller peptides and/or amino acids. Typically, a major portion of the protein will precipitate as a result of the acidification of the plant juice so that it can be removed by conventional means, e.g. centrifugation.
• the green crop drying industry produces fodder pellets by drying crops such as perennial rye grass (Lolium perenne), Italian rye grass (Lolium multiflorum), clover grass and alfalfa
• crops such as perennial rye grass (Lolium perenne), Italian rye grass (Lolium multiflorum), clover grass and alfalfa
• the crops are steam-heated in a cooker to a temperature of about 80°C This process results in coagulation of a large proportion of the protein, as well as damage to the plant cells
• the crops are pressed, e g using a screw press
• the resulting juice normally contains about 4-6% dry matter
• the dry matter content of the press cake produced in this process is normally about 30-40%, e g about 35%
• vacuum evaporators are used to concentrate some of the juice, and the concentrated brown juice is introduced into the press cake before drying
• the residue from these factories is known as brown juice
• Example 1 shows that sterilisation of brown juice makes it a poor fermentation medium with respect to growth rate, lactic acid yield and the availability of free ammo acids
• Example 1 also shows that fresh green and brown juice are excellent media for lactic acid bacteria, and that lactic acid fermentation of the fresh juice increases the value of the juice for fermentation by bacteria requiring free ammo acids
• Example 1 also shows that if the fresh juice is inoculated with a pre-culture of lactic acid bacteria, for example a culture of Lactobacillus delbruecku, the result is an excellent growth medium which in fact is superior to traditional complete growth media for lactic acid
• Plant juice can be obtained from any suitable fresh crop, for example Italian rye grass, alfalfa or clover (green juice) and various heated green crops (brown juice), as well as from e g Jerusalem artichoke stems and tubers, sugar beet tops or potato tubers
• the non-ste ⁇ lised juice is inoculated with a pre-culture of lactic acid-producing bacteria
• lactic acid-producing bacterium/bacteria includes in particular non- sporeforming, mesophihc or anaerobic rod-shaped or coccoid non-sporeformmg bacteria having in common the ability to produce lactic acid
• the bacteria will be of the genus Lactobacillus, such as L helveticus, L delbruecku, L casei, L acidophilus, L bulga ⁇ cus , L plantarum, L paracasei spp paracasei or L salivanus
• Thermophilic lactic acid-forming bacteria such as Bacillus stearothermophilus or Streptococcus thermophilus can also be used
• the microorganism used for acidifying the juice will typically be a homofermentative (i e producing only lactic acid as a fermentation product), mesophilic lactic acid bacterium with a growth optimum between about 30°C and about 50°C or a homofermentative, thermophilic bacterium with a growth optimum between about 50°C and about 70°C
• the temperature of the plant juice being subjected to fermentation will of course be adjusted in each individual case according to the requirements of the bacteria culture in question
• the initial fermentation is continued until the pH drops to below about 4 5, and preferably to below about 4 0, e g to a pH of between about 3 5 and 4 0
• the resulting lactic acid fermented juice is a stable product that can easily be stored under anaerobic conditions for further use as a fermentation medium, ei- ther as is or in concentrated form
• Example 2 it is shown that if the non-concentrated, fermented juice is kept in a cistern at room temperature it can be stored for at least 6 months without loss of bio-available ammo acids
• the acidified, fer- mented plant juice can be concentrated, for example using conventional evaporation techniques known in the art A preferred technique for this purpose is vacuum evaporation
• the concentrated, acidified juice can be used as such for further fermentations or it can be diluted, e g with non-concentrated, acidified juice
• the acidified juice When the acidified juice is concentrated, it will typically have a lactic acid concentration of about 15-40% (w/v), more typically about 20-30%, e g about 25% Juice which has been acidified by means of addition of acid can of course also be concentrated using similar techniques
• the acidified plant juice is used as a fermentation medium, for example in one or more of the fermentation processes described below
• these fermentations can be performed as either a batch, fed-batch or continuous process
• the result of the method of the invention is a fermented mixture containing the microorganisms used for the fermentation as well as the fermentation product produced by the microorganisms in question
• the invention provides the advantage that all of the dissolved components of the fermentation mixture are turned into useful products, thus eliminating new waste products as well as any need for further purification steps
• the invention it is possible to turn otherwise useless waste products that represent an economically and environmentally expensive problem into valuable end products
• the invention solves an environmental problem that until now has remained unsolved due to the lack of an economically satisfactory solution
• water will simply be removed from the fermentation mixture by conventional means such as evaporation to result in a final product containing dry matter originally present in the mixture as both suspended matter such as microorganism cells and dissolved compounds
• the biomass may be separated from the liquid by means of e g centrifugation and/or filtration, and the supernatant may then be dried by means of e g evaporation and/or spray drying
• all suspended and dissolved matter in the fermentation mixture end up as useful products
• the acidified fresh green or brown juice is typically supplemented with additional carbohydrates, so that the initial acidification step using e g lactic acid fermentation (for preservation of the juice) is followed by an additional lactic acid fermentation step
• Any suitable carbohydrate source can be used for this purpose
• the carbohydrate source will typically be an inexpensive and readily available material with a high carbon content, for example sugar beet molasses
• the acidified juice can be inoculated with additional lactic acid bacteria
• the acidified green or brown juice serves as a complete medium for the lactic acid bacteria, i e it is normally only necessary to add a carbon source Addition of other nutrients, on the other hand, is normally unnecessary, since these other nutrients will typically be found in appropriate quantities in the acidified juice
• the acidified juice to be used for the actual lactic acid production can be in either non-concentrated or concentrated form, or in the form of a suitable mixture.
• Example 3 The production of lactic acid in a fermentation medium produced according to the invention, without any addition of nutrients to the juice, is illustrated in Example 3.
• Example 4 shows that a high concentration of lactic acid can be obtained using acidified plant juice according to the invention supplemented only with molasses as a carbon source.
• lactic acid is intended to refer to any one of these types of lactic acid or mixture thereof.
• lactic acid When the fermentation broth has obtained the desired maximal concentration of lactic acid, e.g. about 100 g/l, addition of additional carbohydrates is discontinued, and the fermentation ceases.
• any suitable conventional separation technology such as centrifugation, evaporation, ultrafiltration or electrodialysis, or a combination thereof, may be used.
• the resulting lactic acid can be used in a known manner e.g. in the food industry or for the production of biodegradable plastics such as poly-lactic acid.
• the lactic acid produced as described above can further be used for the production of acetic acid or salts thereof.
• the product of the additional lactic acid fermentation step optionally in concentrated form, e.g. having a concentration of lactic acid of at least about 10% (w/v)
• a suitable culture of acetic acid-producing bacteria Clostridium formiaceticum is an example of such an acetic acid-producing bacteria.
• the lactic acid produced by the lactic acid bacteria will be the carbon source, and acetic acid will be the end product. Production of acetic acid in this manner is described in Example 5. Further fermentation: Production of amino acids and amino acid salts
• the present invention also has a great potential for the production of amino acids by bacteria belonging e.g. to the Corynebacterium group.
• acidified juice com- prising lactic acid typically with a lactic acid concentration of at least about 2% (w/v)
• carbohydrate source and a nitrogen source and inoculated with amino acid producing bacteria in order to produce amino acids.
• a fermentation medium produced e.g. from brown juice according to the present invention contains nearly all the chemical compounds needed for the amino acid fermentation in readily available form; only ammonium sulphate and additional carbohydrate have to be added to the medium in this case.
• Example 6 illustrates L-lysine fermentation based on deproteinised evaporated potato juice.
• brown juice is used for high-yield L-lysine production.
• lactic acid-fermented juice prepared according to the present invention in contrast, with lactic acid-fermented juice prepared according to the present invention, a very high growth rate together with a very high yield and productivity is obtained. It has thus been found that the acidified juice resulting from the initial lactic acid fermentation as described above is a perfect substrate for amino acid-producing microorganisms, which are able to utilise the available organic acids and amino acids as building blocks and as an energy source for production of both cell mass and the desired amino acids, for example L-lysine.
• Example 8 shows that use of the acidified plant juice makes possible a very high microorganism growth rate as well as a very high yield and productivity.
• the biomass yield is increased in the lactic acid- fermented juice, as the lactic acid bacteria are able to utilise a higher proportion of the organic acids and sugars present in the juice than is the case for the Corynebacteria alone.
• the lactic acid bacteria hydrolyse some of the proteins in the juice to free amino acids and bio-available peptides, thereby making them available for the Corynebacteria in the subsequent amino acid fermentation. The Corynebacteria are therefore able to utilise the produced lactic acid for both cell mass and amino acid production.
• the net result is that the lactic acid fermentation converts compounds which are otherwise non-fermentable for the Corynebacteria to compounds which the Corynebactena are able to ferment.
• the overall yield is therefore greatly increased. Significantly, this takes place without removing any vital compounds.
• L-lysine lactate is a much more interesting compound as both parts can be utilised in the feeding of animals, L-lysine for protein synthesis and lactic acid as a carbon source and as probiotic that prevents intestinal infections.
• Such a fermentation process is shown in Example 9. In this example, half of the needed amount of ammonia is added at the beginning of the fermentation, the rest being added as pH control in order to keep the pH at about 7.0.
• the amount of added ammonia is furthermore controlled by a computer model showing the relationship between added ammonia and fermentation time.
• the lactic acid concentration in the medium is controlled by adding more fermented, evaporated brown juice as necessary, the relative amounts of added ammonia and acidified brown juice being chosen so as to maintain the pH at about 7.0.
• the end product e.g. lysine lactate feed concentrate or crystalline lysine lactate
• a product such as a lysine lactate feed concentrate will have a duel function in that addition of L-lysine optimises protein synthesis while lactic acid is used as an energy source as well as a probiotic.
• the present invention provides a much less expensive method for producing fermentation media than prior art methods, and in addition it solves an environmental problem for green crop dryers and other agro-industrial plants having plant juice as a waste material.
• the resulting fermentation medium is a complete medium with a high content of free amino acids that is highly useful for microorganisms able to utilise lactic acid as a carbon source. Additives such as corn steep liquor, yeast extract and other sources of amino acids and vitamins normally used in fermentation media are therefore unnecessary. Since the carbohydrates in the fresh juice are converted to lactic acid during the initial lactic acid fermentation, the acidified juice can easily be stored before possible evaporation and sterilisation. A further advantage is that the acidified juice can be sterilised without damage caused by Maillard reactions between carbohydrates and amino acids.
• Fig. 1 is a flow sheet illustrating an example of the method of the invention for the production of useful products based on agricultural waste products.
• compounds for use e.g. as a functional feed are produced based on plant juice from green plants such as grass as well as from juice from potatoes.
• the fermentation medium is a mixture of the treated green plant juice and potato juice supplemented with a carbohydrate source, and the fermented mixture is evaporated to result in any of a number of different fermentation products (for example calcium magnesium acetate, lysine feed concentrate, lysine lactate feed concentrate, ammonium lactate feed concentrate) without resulting in any new waste products.
• Plant juice was prepared from fresh Italian rye grass, alfalfa and clover (green juice) and from heated green crops (brown juice) as well as from Jerusalem artichoke stems, sugar beet tops and potato tubers.
• Fresh brown juice derived from a mixture of crops was either inoculated, without sterilisa- tion, with a pre-culture of one of the three different lactic acid bacteria (L. salivarius, L. delbruecku or L. paracasei) or was sterilised at 121 °C for 30 minutes and, after cooling to fermentation temperature, inoculated with the pre-cultures. Fermentation was carried out for different periods of time at a temperature of 40°C, and the lactic acid yield based on the amount of sugar in the juice was determined. The results are provided in Table 1 below.
• Table 2 shows that the lactic acid fermentation of the non-sterilised juice results in a content of available free ammo acids that is up to several times higher than the content in either the raw juice or the fermented, sterilised juice
• Table 3 shows the content of ⁇ -ammo N, L-threonine and L-methionine, in mg/l, in lactic acid fermented juice from alfalfa stored for over a year at room temperature It can be seen that the juice can be stored for up to 9 months without any significant loss of essential ammo acids
• Table 5 shows that almost all of the organic acids originally present in non-sterile brown juice subjected to lactic acid fermentation were consumed in the fermentation pro- cess, i e the organic acids were converted to lactic acid
• Example 4 Further lactic acid fermentation in acidified green or brown juice supplemented with molasses
• Brown juice from a mixture of green crops was preserved by fermentation with lactic acid bacteria until the pH of the juice fell to about 4.0 as described in Example 2. After storage under anaerobic conditions for 2 months, the acidified juice was supplemented with molasses from sugar beets or sugar cane. The pH was adjusted to 6.25, and the fermentation continued until all of the added sugars were converted to lactic acid. Table 6 below shows the yield of lactic acid based on the amount of added sugar from molasses.
• Table 6 shows that the yield based on added sugars is very high, and that the fermentation requires no supplementary addition of growth factors.
• Lactic acid fermentation may be performed as described in Example 4. After this fermen- tation, the lactic acid containing medium is sterilised and a pre-culture of Clostridium for- miaceticum is added. Fermentation to produce lactic acid takes place under anaerobic conditions. Since the fermentation medium contains all the necessary growth factors for this strain of acetic acid-producing bacteria as well as lactic acid as a carbon source, the lactic acid in the medium is converted to acetic acid. If the resulting acetic acid is neutralised using dolomite lime and concentrated by evaporation, a calcium magnesium acetate (CMA) product useful as a de-icing compound is obtained
• the composition of the fermentation medium was
• 0.3 I of seed culture was inoculated into 2.7 I of the above-mentioned culture medium in a 5 I fermentor. Cultivation was carried out at 30°C for 50 hours with aeration at a rate of 5 l/min and agitation at between 500 and 1250 rpm. During the cultivation, the pH was kept at 6.8 by addition of L-lactic acid and ammonia in a 25% aqueous solution. After completion of the fermentation, the resulting culture liquid contained 47 g L-lysine per litre. The culture medium was acidified and evaporated as described in Example 6.
• green juice and brown juice from different sources are used for the production of a lysine feed concentrate.
• a fermentation medium was prepared using supernatant from green juice from alfalfa that had been fermented with Lactobacillus salivarius BC1001 in a continuous process at 40°C, together with brown juice from a mixture of crops that had been fermented with Bacillus stearothermophilus in a continuous process at 60°C and then vacuum evaporated to a dry matter content of 35% (w/v).
• the composition of the fermen- tation medium was as follows: all ttaZ!
• Example 9 Production of lysine lactate feed concentrate on the basis of acidified green juice, concentrated acidified brown juice and molasses
• This example illustrates the production of a lysine product containing lysine with lactic acid as the counter ion
• the same media and microorganisms as in Example 8 were used, except that sulphuric acid and ammonium sulphate were omitted
• composition of the medium was as follows

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