METHOD FOR TREATING ORGANIC WASTE PRODUCTS

Abstract: A method for treating an organic waste product comprising plant juice, by providing an acidified and deproteinised plant juice having a pH of less than about 4.5 and 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. Acidification may be performed by addition of an inorganic or organic acid, or preferaby, by inoculating plant juice with a culture of lactic acid-producing micro-organisms and incubating the inoculated plant juice at a suitable temperature until an acidified and storage-stable plant juice product having a pH of less than about 4.5 is obtained. The fermentation product may be an organic acid such as lactic acid, and amino acid, a feed additive containing both lactic acid and an amino acid, an enzyme, protein or peptide, or a fungus or bacteria.

Description: METHOD FOR TREATING ORGANIC WASTE PRODUCTS Field of the invention 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. Background of the invention 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. Despite improvements that have been made in recent years, such technologies still suffer from unsolved waste problems, for example liquid waste in the form of plant juice. An in- creasing awareness of and focus on these problems, together with new legislative restrictions placed on e.g. disposal of plant juice, makes it important to find new ways of dealing with such waste material, preferably by re-using as much of the waste material as possible for the production of new, useful products. In addition, future demands on bio-based productions will be economical as well as environmental sustainability. These demands will in turn result in demands on the crop production methods, including management of crop waste products. For reasons of energy, environment and economy it will be necessary for crops to fit well into the normal crop rotation scheme, and there will be an emphasis on having a substantial net energy surplus from primary production and from conversion in the bio-refinery, as well as an emphasis on having few or preferably no waste products that can present environmental problems. In the temperate zone, 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. For the production of potato starch, 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. However, due to the environmental problems involved in applying excess nitrogen, which if not taken up by plants in the autumn and winter period will eventually end up in the ground water as nitrate, more and more restrictions have been introduced regarding the use of these residues as fertilisers. For example, in the Netherlands there is a total ban on using plant juice as fertiliser, while in Denmark there is a ban on applying potato and grass juice as a fertiliser from October 1 st to February 1 st . 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 M Andersen and P Kiel ("Agricultural residues and cereals as fermentation media", in Cereals Novel Uses and Processes, edited by Campbell et al , 1997) describe the use of green juice as a fermentation medium to produce organic chemicals Green juice resulting from wet separation of green crops was subjected to a primary lactic acid fermentation to result in a storage-stable fermentation product suitable for L-lysine fermentation Brief disclosure of the invention 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 Significantly, 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 Further, 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 In a particular embodiment, the invention may be used for producing functional feed or a functional feed additive The term "functional feed" refers to feed, e g fodder for non-ruminant livestock or poultry, where one or more components present in the feed or feed additive enhance the digestability of the feed itself and/or that of other feed ingredients with which it has been mixed Such components can e g include enzymes, ammo acids, organic acids, microorganisms, etc In addition to use as an animal feed or animal feed additive, the fermentation products produced according to the invention may be used for various industrial purposes Examples of such 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 ) 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. As a result, addition of e.g. amino acids, vitamins, minerals, etc. is normally unnecessary. And since essentially all of the organic and inorganic compounds are utilised in the fermentation process, no waste product containing such compounds is produced. 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. In its broadest aspect, the invention involves pre-treatment of plant juice to provide an acidified and deproteinised plant juice which is subsequently used as a fermentation medium. In a preferred embodiment, 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, however, is to obtain acidification by adding an acid to the plant juice, followed typically by heating, centrifugation and removal of protein. After this pre-treatment to obtain an acidified and deproteinised plant juice, 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. Detailed description of the invention In one aspect, 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 As used herein, the term "plant juice" is intended to encompass any juice obtained from any suitable plant or part thereof In particular, 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 As will be apparent from the discussion below, fresh juice includes both "green juice" obtained by pressing fresh plant biomass as well as "brown juice" obtained by heat-treatment of plant biomass It will also be clear that 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 It will be apparent from the above that 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 Further, since the invention is directed to plant juice that exists as a waste product, 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 Thus, it will be understood that 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 Similarly, 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%. According to the invention, fresh plant biomass, for example grass, clover or alfalfa, or biomass that has been stored for a short time in the field, e.g. a few days, such as one to two days, is cut and pressed, and the juice obtained from the pressing is preferably acidified by lactic acid bacteria. As a result, several important advantages are obtained: • a stable product with a pH of typically between 3.5 and 4.5 is formed, • the carbohydrates in the juice are converted to organic acids, especially lactic acid, • a part of the proteins are precipitated, • the rest of the proteins are partly hydrolysed to readily available free amino acids, and • vitamins in the juice are kept unspoiled. As used herein, 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. As indicated above, a preferred method for obtaining acidified plant juice is by means of lactic acid fermentation, since this method is easy, inexpensive and efficient. If desired, however, 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. The term "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. It will be understood from the discussion herein that the acidified and deproteinised plant juice may be used either as is or in concentrated form as the fermentation medium. Green juice 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 In order to reduce the energy consumption in the drying process, some of the water in the grass is normally removed from the green crops before drying in a drum dryer, resulting in a significant production of green plant juice At some green pellet factories pressing is carried out on fresh green biomass, resulting in the production of green juice with a dry matter content of about 3-10% and a press cake with a dry matter content of about 16-22% Brown juice At other green pellet factories, 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 After cooking, 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% In some cases, 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 Using plant juice as fertiliser In the temperate zone, the largest amount of plant juice is typically produced around October-November As explained above, this juice has in the past been applied to fields as a fertiliser, but for environmental reasons this will be increasingly difficult or even impossible in the future Juice produced in this period therefore has to be stored until the spring or used for other purposes However, neither of these options is ideal in connection with prior art techniques Potential for fermentation From silage-making it is known that naturally occurring microorganisms, and especially lactic acid bacteria such as Lactobacillus plantarum, are able to lower the pH and preserve green crops under anaerobic conditions When green juice or brown juice is stored in cisterns or large lagoons before being applied to fields as fertiliser, a natural and uncontrolled fermentation process will very soon take place, and the juice will end up as an unpleasant smelling liquid in which a variety of different microorganisms under uncontrolled conditions convert the organic compounds of the juice to compounds which often have a noxious odour If the juice has to be stored before use in an industrial fermentation process, it is therefore necessary according to the prior art to sterilise the fresh juice by heating it to a temperature of typically 121 °C for about 30 minutes before inoculation with the desired microor- ganism However, the sterilisation procedure spoils the juice as a fermentation medium This is illustrated e g in Example 1, which 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 In contrast to the reduced quality of fermentation media produced using sterilisation, 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 bacte- πa, for example MRS broth Preservation of plant juice by lactic acid fermentation 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 According to this embodiment of the invention, the non-steπlised juice is inoculated with a pre-culture of lactic acid-producing bacteria Any suitable lactic acid-producing bacterium, or a combination of more than one lactic acid bacterium, may be used for this purpose The term "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 Typically, the bacteria will be of the genus Lactobacillus, such as L helveticus, L delbruecku, L casei, L acidophilus, L bulgaπcus , 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 In order to effectively preserve the juice, 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 In 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 If desired, e g for long-term storage or for transportation purposes, 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 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 Following the initial lactic acid fermentation step as described above (or acidification by addition of acid) to result in a storage-stable plant juice suitable for use as a fermentation medium, the acidified plant juice, possibly after concentration and/or storage is used as a fermentation medium, for example in one or more of the fermentation processes described below It will be clear to persons skilled in the art that 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 By means of the invention it is possible to turn otherwise useless waste products that represent an economically and environmentally expensive problem into valuable end products In other words, the invention solves an environmental problem that until now has remained unsolved due to the lack of an economically satisfactory solution Often, for example for use as a feed additive, 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 In other cases, for example when the fermentation product is a chemical to be used for industrial purposes, the biomass (suspended matter) 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 In both cases, all suspended and dissolved matter in the fermentation mixture end up as useful products Further fermentation Production of lactic acid If the acidified juice is to be used for an actual lactic acid production, 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 For practical and economic reasons, the carbohydrate source will typically be an inexpensive and readily available material with a high carbon content, for example sugar beet molasses If necessary or desired, the acidified juice can be inoculated with additional lactic acid bacteria When used for the actual production of lactic acid, 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. When performing such an additional lactic acid fermentation step, the pH of the fermentation medium may be adjusted as necessary to the optimal pH for the lactic acid bacteria in question by suitable addition of an organic or inorganic base. 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. By using different strains of lactic acid bacteria, it is possible to form the L(+), L(-) or D(-) forms of lactic acid, as well as mixtures of L(+)/(-) and D(-). As used herein, the term "lactic acid" is intended to refer to any one of these types of lactic acid or mixture thereof. 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. For purification of the lactic acid, 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. Further fermentation: Production of acetic acid and salts thereof The lactic acid produced as described above can further be used for the production of acetic acid or salts thereof. In this case, 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), is sterilised, after which it is inoculated with a suitable culture of acetic acid-producing bacteria. Clostridium formiaceticum is an example of such an acetic acid-producing bacteria. In this process, 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. In this case, acidified juice com- prising lactic acid, typically with a lactic acid concentration of at least about 2% (w/v), is sterilised and subsequently combined with a carbohydrate source and a nitrogen source and inoculated with amino acid producing bacteria in order to produce amino acids. Compared with, for example, a typical synthetic medium for L-lysine fermentation by a member of the Corynebacterium group such as C. glutamicum, 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. In Example 7, brown juice is used for high-yield L-lysine production. Although it is known that untreated plant juice can be used as fermentation medium for the production of amino acids, the result using prior art methods is a low yield of cell mass and amino acids. Sterilised plant juice, on the other hand, contains only a relatively small amount (about 30% of the dry matter) of sugars that are fermentable by Corynebacteria and only a limited amount of free amino acids. If untreated juice is pre-treated as disclosed in WO 92/19716, a higher yield is achieved, but as mentioned above, the method of WO 92/19716 is expensive and requires a large energy expenditure. Furthermore, the method of WO 92/19716 does not allow the juice to be stored before sterilisation. 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. In particular, it has been found that 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. Also, 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. If the process for producing amino acids is controlled with respect to ammonia and lactic acid, it is possible to exchange ammonium sulphate or ammonium chloride with ammonia and lactic acid and thereby end up with a product containing lactate as a counter-ion to L- lysine instead of sulphate or chloride. 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 resulting product has three important advantages: 1. The end product, e.g. lysine lactate feed concentrate or crystalline lysine lactate, will only add valuable organic compounds to the feed and not unwanted elements or groups such as chloride and sulphate which have no value for the animals and which represent a potential environmental problem when manure from the animals is used as fertiliser. 2. 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. 3. Removal of sulphate from the fermentation medium will reduce crystallisation prob- lems when evaporating the fermentation medium to obtain a lysine feed concentrate. In summary, 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. Description of the drawing 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. In this case, 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. The invention will be further illustrated by the following non-limiting examples- Example 1 Preservation of plant juice by lactic acid fermentation 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 1. Lactic acid yield for sterilised and non-sterilised brown juice During lactic acid fermentation, part of the protein in the juice is hydrolysed, so that the content of free amino acids in the juice increases. In Table 2, it is shown that the non-sterilised green juice after lactic acid fermentation for a period of 5 hours at a temperature of 40°C has become a more valuable product for further fermentation by bacteria that require amino acids, while the juice that was sterilised before inoculation has not been im- proved. Table 2 Content, in mg/l, of the L-form of some free ammo acids in fresh green juice, and in the same juice, sterilised or non-steπhsed, after fermentation with the lactic acid bacterium Lactobacillus salivanus for 5 hours at 40°C For comparison purposes, the ammo acid amounts are adjusted to correspond to a dry matter content of the juice of 10% 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 Example 2 Stability of stored lactic acid fermented green juice Table 3 below shows the content of α-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 3: Nitrogen/ammo acid content in stored, lactic acid-fermented juice SUBSTITUTE SHEET pUtf 26) Example 3 Production of lactic acid from green and brown juice Green juice from clover and alfalfa, and brown juice from a mixture of crops were heated to a temperature of 40°C and inoculated with a 5% inoculum (50 ml per litre of fermentation medium) of L delbruecku As shown in Table 4 below, a very high yield of lactic acid was reached, i e greater than 100% of the sugar present in the juice This is possible because not only the sugars, but also some of the organic acids in the juice, are used for growth and lactic acid production Table 4. Yield of lactic acid by inoculation of green juice and brown juice with L delbruecku Table 5 below 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 Table 5 Content in g/l of some organic acids before ("substrate") and after lactic acid fermentation of non-sterile brown juice with lactic acid bacteria using (1) L delbruecku, (2) L salivanus or (3) L paracasei ssp paracasei 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. Lactic acid fermentation of preserved brown juice to which cane molasses was added in an amount of 23.21 g/l. The fermentation was carried out with the lactic acid bacteria L. delbruecku, L. salivarius and L. paracasei ssp. paracasei. Table 6 shows that the yield based on added sugars is very high, and that the fermentation requires no supplementary addition of growth factors. Example 5 Production of acetic acid and calcium magnesium acetate (CMA) by a two-stage fermentation of deproteinised potato juice or brown juice supplemented with molasses 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 Example 6 Production of lysine feed concentrate on the basis of evaporated, deproteinised potato juice and sugar beet molasses This example illustrates the use of deproteinised and evaporated potato juice (EDPJ) for the production of an L-lysine feed concentrate The corynebacteria Corynebacterium glu- tamicum strain V5 ATTC 700239 was used for this experiment The composition of the fermentation medium was g/l il EDPJ with a dry matter content of 54 2% 150 Ammonium sulphate 25 D-glucose 90 Biotin 150 Thiamin 200 0 5 I of seed culture was inoculated into 50 I of the above-mentioned medium in a 100 I fermentor Cultivation was carried out at 30°C for 52 hours with aeration at a rate of 50 l/min and stirring at 500 rpm During the cultivation, the pH in the medium was maintained at 6 8 with ammonia gas After completion of the cultivation, the resulting culture liquid contained 35 g of L-lysine per litre The culture medium was then acidified with concentrated sulphuric acid to pH 4 5 and evaporated to a dry matter content of 60% and an L- lysme content of 125 g/l Example 7 Production of lysine feed concentrate on the basis of concentrated brown juice and molasses This example illustrates the use of concentrated brown juice in an L-lysine fermentation Corynebacterium glutamicum B031 was used for this experiment The fermentation was carried out as a fed batch process with cane molasses added in the last part of the fermentation The composition of the fermentation medium was as follows g/l mil Brown juice, D.S.* = 22.2% 188 MgS0 4 NH 2 0 0.5 Ammonium sulphate 33.3 L-leucin 0.2 Calcium pantothenate 0.01 Thiamine 0.001 Biotin 250 Cane molasses 250 * D.S. = dry matter content (wt.%) 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. Example 8 Production of lysine feed concentrate on the basis of acidified green juice, concentrated acidified brown juice and molasses In this example, green juice and brown juice from different sources (different factories) are used for the production of a lysine feed concentrate. As shown in Examples 2 and 3, it is possible to preserve both brown juice and green juice by lactic acid fermentation and at the same time hydrolyse some of the proteins and peptides to free amino acids and convert all the sugars and most of the organic acids to lactic acid. In this example, 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! Supernatant from lactic acid- fermented green juice, 4 8% D S 400 Fermented, evaporated brown juice, 30 2% D S 100 MgSO 4 7H 2 0 0 5 Ammonium sulphate 33 3 L-leucme 0 2 Calcium pantothenate 0 01 Thiamme 0 001 Biotm 250 Sugar beet molasses 250 0 3 I of seed culture of Corynebacterium glutamicum B031 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 55 g L-lysme per litre The yield was 0 45 g lysine per g glucose The culture medium was acidified with sulphuric acid and evaporated as described in Example 6 in order to obtain the final lysine feed concentrate product 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 The composition of the medium was as follows q/l t- aZ! Supernatant from lactic acid- fermented green juice, 4 8% D S 400 Fermented, evaporated brown juice, 30 2% D S 200* MgSO 4 7H 2 O 0 5 Ammonia 20* L-leucme 0 2 Calcium pantothenate 0 01 Thiamme 0 001 Biotm 250 Sugar beet molasses 200 * Initial amount Brown juice and ammonia were added ad libitum during the fermentation to control pH 0 3 I of seed culture of Corynebacterium glutamicum B031 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 the lactic acid fermented, evaporated brown juice and ammonia in a 25% aqueous solution After completion of the fermentation, the resulting culture liquid contained 65 g L-lysine lactate per litre The yield per g sugar was 0 45 g lysine lactate The culture medium was acidified by stopping the air supply and the pH regulation when the sugar content in the medium reached 10 g/l Fermentation was stopped when the pH reached 4 5 The fermentation product was then pasteurised and evaporated as described in Example 6 in order to obtain the final lysine lactate feed concentrate product with an L-lysme lactate content of 20% by weight

Claims: Claims 1 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 2 The method of claim 1 , wherein acidification is performed by inoculating plant juice with a culture of lactic acid-producing microorganisms and subjecting the inoculated plant juice to a first fermentation step at a suitable temperature until acidified plant juice having a pH of less than about 4 5 is obtained 3 The method of claim 1 , wherein acidification is performed by adding to the plant juice at least one inorganic or organic acid to result in a pH of less than about 4 5 4 The method of any of claims 1-3, wherein the acidified plant juice has a pH of below about 4 0 5 The method of any of claims 1-4, wherein the acidified and deproteinised juice is stored under anaerobic conditions at a temperature of less than about 20°C, preferably less than about 15°C, until it is used as a fermentation medium 6 The method of claim 2, wherein the acidified and deproteinised juice is concentrated, e g by vacuum evaporation, e g to a lactic acid concentration of at least about 15% (w/v), such as about 20-30% 7 The method of any of the preceding claims, wherein the acidified and deproteinised juice, optionally in concentrated form, is used as a fermentation medium for the production of lactic acid by adding carbohydrates as necessary to the acidified juice so as to maintain lactic acid fermentation until a desired lactic acid concentration is obtained 8 The method of any of the preceding claims, wherein acidified and deproteinised juice comprising lactic acid is sterilised and subsequently inoculated with acetic acid producing bacteria in order to convert lactic acid to acetic acid 5 9 The method of any of the preceding claims, wherein acidification and further fermentation is performed substantially without addition of additives other than carbohydrate sources to the fermentation medium 10 The method of any of claims 1-7, wherein acidified and deproteinised juice having a 10 lactic acid concentration of at least about 2% (w/v) is sterilised and subsequently combined with a carbohydrate source and a nitrogen source, e g ammonia, and inoculated with ammo acid producing bacteria in order to produce ammo acids 11 The method of claim 10, wherein a fermentation broth containing L-lysme and organic 15 acids is concentrated by e g vacuum evaporation to a lysine feed concentrate or lysine lactate feed concentrate suitable for use as an additive in animal feed 12 The method of claim 2, wherein the microorganisms used for acidifying the juice are homofermentative, mesophilic lactic acid bacteria with a growth optimum between about 20 30°C and about 50°C or homofermentative, thermophilic bacteria with a growth optimum between about 50°C and about 70°C 13 The method of any of the preceding claims, wherein the fermentation product is selected from the group consisting of 1) organic acids, 2) ammo acids, 3) feed additives 25 containing both lactic acid and an ammo acid, 4) enzymes, proteins and peptides, and 5) yeasts, moulds and other fungi as well as bacteria 14 A fermentation product produced by the method of any of claims 1-13 30 15 An animal feed or feed additive comprising a fermentation product according to claim 14

Inventors: ANDERSEN MARGRETHE, KIEL PAULI, ANDERSEN, MARGRETHE, KIEL, PAULI
Applicants: AGRO FERM AS, ANDERSEN MARGRETHE, KIEL PAULI, AGRO-FERM A/S, ANDERSEN, MARGRETHE, KIEL, PAULI
Application year: 2000
Main classification: C12P7
IPC codes: C12P7, C12P13