FIELD OF THE INVENTION

[0001] The present invention relates to a sustainable biorefinery concept and method for cost-efficient utilization industrial by-products and conversion into valuable end products.

BACKGROUND OF THE INVENTION

[0002] Expanding industrialization and increasing populations around the world continues to create an ever-increasing demand for energy, food, and potable water. These trends, coupled with the need to create a sustainable environment, have pushed to recover various industrial by-products for use as animal feed, such pig feed. However, diarrhea is a common problem among swine and piglets. Especially after weaning (at the age of 3-5 weeks) piglets' resistance to pathogens is at its weakest and therefore it is easy for harmful intestinal pathogens to colonize the small intestine causing diarrhea. Diarrhea causes suffering to animals, weakening of productivity resulting from the deceleration of growth and can even lead to deaths of piglets.

[0003] In modern pork production, many farms have specialized in meat production and buy piglets outside. Thus, piglets that have been born in different environments and are used to different types of microbial populations are brought into the same unit with yet different microbial population. The changing microbiological environments, transports, reorganizations of pigs, and changes in feeding exposes animals to diarrhea. Furthermore, pork production in large production units makes it even harder to control the spreading of diseases.

[0004] Unlike ruminants pigs lack the abilities to use fibres as a source of energy and to produce all the amino acids they need. Therefore pigs are quite demanding regarding the composition of their feedstuffs. Several food industry by-products have, however, been found to be suitable for use as feedstuff for pigs. Many of the food industry by-products that may be suitable for use as feedstuffs for pigs are in liquid form. Liquid food industry by-products can be obtained e.g. from brewing and distilling industries, starch industry, dairy industry, potato processing, and sugar industry (Pedersen et al. 2005, Scholten et al. 1999). Several million tonnes of liquid co-products from food industry, especially liquid wheat starch, potato steam peel, and cheese whey rich in sugar and protein, are annually recycled as pig nutrition in Europe (Scholten et al. 1999). However, owing to the nature of such food industry byproducts, spoilage is a general problem associated thereto.

[0005] Important basic factors in the prevention of diarrheal diseases include high level of hygiene, farm conditions, and feed composition. In addition, antibiotics have been used as additives in feed to improve the health and well-being of animals and to promote the growth, especially in piglet and poultry industries. However, the use of most antibiotics as growth-promoters has been banned in the EU. Therefore, today antibiotics are only used to prevent and treat microbial infections. Antibiotics may be administered either individually as injections or as group medications mixed in feed or drink. Medicating individuals is laborious whereas group medications are easier and more efficient to perform. A general problem associated with extensive use of antibiotics is the generation of resistant microbial strains.

[0006] Zinc oxide has also been used in the prevention of diarrhea in weanling pigs. Zinc oxide not only prevents diarrhea but also stimulates growth rate and feed intake in pigs when included in the diet at levels considerably higher than the nutritional requirement for zinc. The mechanism that is involved in the action of pharmacological levels of zinc is not well understood. However, it is thought to be somewhat like the action of antibiotics in that it modifies the bacterial population in the gastrointestinal tract. Anyhow, zinc is a heavy metal and, thus, its use in food production may be questionable.

[0007] The production of pig feeds containing medicinal additives has more than quadrupled from year 2000 (924 000 kg) to year 2010 (4 200 000 kg) (Evira 201 1 ). The production has risen mainly due increase in the amount of medicinal porcine feed. About ¾ of these porcine feeds contain zinc oxide as active ingredient (MMM 2010, p. 23). In 2010 zinc oxide covered 97 % of the active medicinal ingredients used in pig feeds produced in Finland (Evira 201 1 ).

[0008] There is thus an identified need for sustainable methods of producing pig feed that is safe and enhances the wellbeing of pigs and piglets.

BRIEF DESCRIPTION OF THE INVENTION

[0009] An object of the present invention is to provide a biorefinery, wherein biomass is used as a starting material and converted by lactic acid fermentation into fermented solids and evaporable agents. [0010] Another object of the present invention is to provide a method of converting biomass into fermented solids and evaporable agents by lactic acid fermentation. Said method comprises the steps of a) heating the biomass to a temperature of about 55°C to about 75°C; b) inoculating said biomass with lactic acid bacteria; c) fermenting the biomass at about 27°C to about 37°C in a pH of about 3.5 to about 5; d) performing vacuum distillation at about 45°C to about 55°C under a vacuum of about 1 bar; and e) recovering fermented solids and distilled evaporable agents.

[0011] Still another object of the present invention is to provide a food product obtainable through the biorefinery or conversion method set forth herein. In some embodiments, the food product an animal feed, such as a pig feed.

[0012] According to some embodiments of the present refinery and conversion method, the lactic acid fermentation is initiated by an inoculumn comprising lactic acid bacteria selected from the group consisting of Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus curvatus/sakei, Lactobacillus kefiri, Leuconostoc citreum, Pediococcus pentosaceus, and any combination thereof.

[0013] Other objects, embodiments, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In the following the invention will be described in greater detail by means of preferred embodiments with reference to the attached drawings, in which

[0015] Figure 1 is a schematic representation of samples taken from a malting process.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention is based on developing and elaborating a new type of a biorefinery concept that utilizes industrial by-products, especially from food industry, as raw materials for cost-efficient production of at least two different types of end-products that may be separated from each other on the basis of their physical properties.

[0017] Accordingly, the present invention provides a biorefinery and a biorefining process for converting biomass, preferably low-value biomass, to fermented value-added biomass and evaporable material. The whole concept has been designed to be sustainable, i.e. capable of being continued with minimal long-term effect on the environment, and be operable with industrial waste energy.

[0018] Herein, the term "biorefinery" means a sustainable processing of biomass into a spectrum of marketable bio-based products and bio- energy, according to the definition suggested by International Energy Agency (IEA). To produce these bio-products, biorefineries can apply many hybrid technologies from different fields, such as bioengineering, polymer chemistry, food science, and agriculture.

[0019] In accordance with the above definition, the present biorefinery concept utilizes biomass as a starting material. As used herein, the term "biomass" refers to a biological material obtained directly or indirectly from living organisms. In other words, biomass means any carbohydrate-containing, i.e. organic, material derived from animals or plants. Non-limiting examples of suitable biomass sources for use in the present embodiments include byproducts of food industry, such as cull potatoes, by-products of brewing industry, such as spent grains (e.g. from malt, wheat, or barley), spent hops, mash, and surplus yeast, by-products of dairy industry, such as whey (possibly contaminated with vegetable oils) and effluents consisting of eluted milk and whey, and by-products of sugar industry, such as molasses. In some other embodiments the starting material may be, for instance, a chemically processed or otherwise demanding biomass, such as pulp or bark residue. As an alternative to the above-mentioned low-value biomasses, the present invention may in some embodiments utilize primary starting materials such as triticale or oat.

[0020] A first step of the present biorefining process comprises heating in order to destroy at least a majority of microorganisms in the starting material, which might interfere with a fermenting process or contaminate the end product. A preferred temperature range for heating is between 55°C and 75°C since it is high enough for destroying or inhibiting the growth of any unwanted microorganisms but low enough to enable utilization of industrial waste energy, such as low-temperature waste heat. Duration of the heating step depends on different variables such the nature and amount of microorganisms in the starting material. A person skilled in the art can determine said duration easily.

[0021] As used herein, the term "low-temperature waste heat" refers to surplus heat recovered e.g. from condensing power stations, nuclear power plants or various industrial processes, such as brewing. Low-temperature waste heat may also be recovered from district-heating return pipelines.

[0022] Next step of the biorefining process is balancing of the fermentation conditions. This step is fully optional and depends on different variables such as the quality and quantity of the starting material. Typically, this step, if present, comprises adding nutrients, e.g. in the form of whey, the required amount of which may be easily calculated by a person skilled in the art. Typically, the fermentation process is powered by a sugar content of 0.5% in the biomass to be fermented. Thus, this value can be achieved, for instance, by adding whey having a lactose content of 5% to the biomass to be fermented in a proportion of 1 :10 (v/v).

[0023] In addition, the balancing step may comprise incorporating enzymes into the starting material, such as enzymes which degrade carbohydrates. Typical enzymes suitable for this purpose include various cellulolytic, hemicellulolytic, proteolytic, and amylolytic enzymes.

[0024] In the next step, the optionally balanced biomass is inoculated with lactic acid bacteria (LAB) for lactic acid fermentation. As used herein, the term "lactic acid fermentation" refers to biological process by which carbohydrates such as glucose, fructose, and sucrose, are converted into lactic acid.

[0025] LAB are a group of gram-positive, anaerobic, micro- aerophilic or aerotolerant, catalase-negative rods or cocci capable of lactic acid fermentation. LAB can be divided into two groups based on the end-products of carbohydrate fermentation. Homo-fermentative LAB produce only lactic acid, while hetero-fermentative LAB produce a mix of lactate, ethanol, CO2 and/or acetate.

[0026] Typically, hetero-fermentative lactic acid bacteria grow in plant-associated environments and ferment hexoses (glucose, fructose, man- nose, and lactose) and pentoses (xylose and ribose) present in plant material. Hetero-fermentative LAB can use several unusual fermentation reactions in addition to classical hetero-fermentation. They can also ferment organic acids, which are commonly present in plant material, and arginine.

[0027] Live lactic acid bacteria remain in the end product. Therefore, it is advantageous to use probiotic LAB in the fermentation process. Herein, the term "probiotic" refers to a live organism which, when administered in adequate numbers, confer a health benefit on the host. It is generally accepted that probiotics affect the host at least by improving its intestinal microbial balance, thus inhibiting pathogens and toxin producing bacteria. Typical examples of probiotic LAB belong to the genera Lactobacillus, Enterococcus, or Bifidobacterium.

[0028] Cereal crop material is not an ideal growth environment for all LAB. Therefore, in connection with the present invention, cereal crops to be used for or collected from a malting process were screened for possible naturally occurring lactic acid bacteria. On the other hand, not all LAB adhere well to pigs' digestive tract. Therefore, pig intestines were isolated and screened for naturally occurring LAB. Generally, bacteria that adhere well to pig intestine grow poorly in a cereal crop based material. Thus, bacterial strains identified from pig intestines were further tested for their ability to grow in mash.

[0029] Cross comparison of the LAB species identified in the cereal crop material and pig intestine revealed Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus cur- vatus/sakei, Lactobacillus kefiri, Leuconostoc citreum, and Pediococcus pento- saceus as ideal species to be used in some embodiments of the present fermentation process. These species or any strains thereof may be used independently or in any desired combination. Importantly, the present biorefinery concept or fermentation method is not limited to the use of these LAB species but is applicable any LAB or combinations thereof as long as they are able to ferment the starting material in question.

[0030] In some specific embodiments, the fermentation process is to be initiated by one of the following alternative combinations of LAB species:

- [0031] Lactobacillus paraplantarum and Lactobacillus reuteri

- [0032] Lactobacillus paraplantarum and Pediococcus pentosaceus

- [0033] Lactobacillus paraplantarum, Lactobacillus reuteri and Leuconostoc citreum

- [0034] Lactobacillus paraplantarum + Pediococcus pentosaceus +

Leuconostoc citreum

[0035] Characteristic to these and other suitable non-limiting combinations is that at least one species has been identified from cereal crop material while at least one other species has been identified from pig intestine. In further specific embodiments, it may be beneficial to include Pediococcus pentosaceus in the initiation of the fermentation process.

[0036] Although the above selection including ideal hetero- fermentive and homo-fermentative bacteria is partly based on identifying pig- intestine-associated LAB, it is envisaged that the same LAB may benefit other animals as well. Non-limiting examples of such animals include poultry and pets. Accordingly, the present fermentation process may be applied in the production of feeds for these animals, as well.

[0037] In some embodiments, one or more pre-cultivations may be required to produce a suitable inoculum with sufficient cell concentration for the main fermentation process. In some cases, the inoculum for the first pre- cultivation may be prepared in a laboratory. A typical inoculation rate ranges from 1 % to 7.5%, and viable count ranges typically from 10 ⁶ to 10 ⁹ cfu/ml.

[0038] After inoculation of LAB and gentle but careful mixing, fermentation is maintained at about 27°C to about 37°C, preferably at about 32°C to about 34°C, more preferably at about 30°C. These temperature ranges allow utilization of industrial or other waste heat for the process.

[0039] During fermentation, pH is maintained below 5, preferably from 3.5 to 5 in order to prevent the growth of any, such as soil bacteria, that have survived the preheating step. Means and methods for monitoring and adjusting pH are readily known to a skilled person.

[0040] Typically but not necessarily, the fermentation is operated as a batch process, the main fermentation being planned to run for about 48 hours. In some embodiments, the estimated time required for the fermentation and cleaning, sterilization, and loading of the main bioreactors during the production of one batch takes about 60 hours. Pre-cultivations and mixing and sterilization of the fermentation medium for the next batch may operate in parallel with the main fermentation process. According to some embodiments, it is envisaged that the facility may operate 320 days per year and therefore 128 batches can be processed annually.

[0041] The fermentation process produces evaporable agents, such as water and organic compounds, as by-products. Non-limiting examples of such evaporable organic compounds include ethanol, acetic acid, propionic acid, butanol, and propanol. These compounds are recovered from the fermented biomass by distillation and may be utilized e.g. as energy or industrial chemicals.

[0042] Distillation is an energy-intensive process which usually takes up a large fraction of the total energy requirement of the whole biorefin- ery. Owing to the need to maintain the lactic acid bacteria viable and to run the process with low-temperature waste heat, high temperatures cannot be used in the distillation. Thus, in some preferred embodiments, evaporable agents are to be separated by vacuum distillation. Typically, vacuum distillation is performed at about 45°C to about 55°C under a vacuum of about 1 bar.

[0043] Ethanol is one of the valuable by-products of the present bio- refining process and its yield depends on many variables, such as the starting material, LAB to be employed, and the duration of the fermentation process. In some embodiments, ethanol production capacity of LAB can be affected though metabolic engineering.

[0044] Ethanol produced in the present biorefinery concept may be delivered to a bioethanol produce for further upgrading. Thus, in some embodiments, the present biorefinery provides a sustainable alternative for the production of bioethanol, as it is formed as a co-product in the process that already uses secondary raw materials. If desired, bioethanol may be used for example to replace fossil fuels in the transport sector.

[0045] After distillation, the fermented biomass may be finalized. This is an optional step and may include different processes, such as drying, removing any odors, and adjusting pH.

[0046] Fermented biomass, or fermented solids, obtainable by the present biorefining process may be used for different purposes. One important application is a fermented food product, such as an animal feed, since organic acids, like lactic acid and acetic acid, are natural preservatives which results in an ideal pH and thus safely inhibits microorganisms that causes food to spoil. Thus, the lactic acid fermentation not only converts biomass into a different foodstuff but it also provides improved microbiological stability and safety of the foodstuff. This is particularly important when waste fractions or cereal-crop- based biomasses, which often contain toxin-producing molds, are used as a starting material. One preferred animal feed obtainable by the present biorefining process is a livestock feed, such as a pig feed.

[0047] Another non-limiting application for the fermented solids is to use it as a soil conditioner in order to improve soil's physical properties, such as water retention, permeability, water infiltration, drainage, aeration and structure. One particularly important purpose is to provide nutrition for plants.

[0048] In some preferred embodiments, the present biorefinery is located such that industrial waste heat may be utilized by the biorefinery. Alternatively or in addition, it may be desirable to locate the biorefinery near ex- isting food industry to avoid transportation costs of biomasses to be used as starting materials.

[0049] It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.

EXAMPLES

Example 1. Isolation of LAB from cereal malting process

[0050] For the isolation of lactic acid bacteria associated with cereal crops, samples were collected from malting process. Samples were enumerated in a way that the ones taken from the seeds before/after washing were denoted by number 1 , the ones taken from the beginning of the germination by number 2, the ones taken from the end of germination by number 3 and the only sample taken from heated (+60°C) seeds after germination by number 4 (Figure 2).

[0051] The samples were homogenized by Stomacher (1 :3 suspension in Ringer-§ solution; 10 g of sample and 20 ml of Ringer) and plated onto MRS plates (Difco) (100 μΙ of dilutions 10°, 10 ^"1 , and 10 ^"2 ) in triplicate. Each plate thereof was then incubated either at 25°C, 30°C, or 35 to 37°C, respectively, in an anaerobic cultivator until bacterial colonies appeared.

[0052] Parallel samples were homogenized by Stomacher (1 :3 suspension in Ringer-§ solution; 10 g of sample and 20 ml of Ringer) and suspended in MRS medium (Difco) (1 ml of samples per 50 ml of MRS) in infusion bottles in triplicate. Each bottle thereof was then incubated either at 25°C, 30°C, or 35 to 37°C, respectively, in an anaerobic cultivator until visible growth appeared. Next, 100 μΙ samples taken from each vial were diluted into Ringer solution (dilutions 10°, 10 ^"1 , and 10 ^"2 ), and plated onto MRS plates. The plates were incubated at respective temperatures until detectable colonies appeared.

Table 1. Number of colonies on each plate

Samplel Samplel Sample2 Sample2 Sample3 Sample3 Sample4 (SR) (SL) (G1 R) (G1 L) (G2R) (G2L) (HGL)

Fresh

samples:

10 ^u (30°C) (3*) TNTC TNTC

10 ^" ' (30°C) 268 TNTC

TNTC; too numerous to count

Example 2. Isolation of LAB from pig intestine

[0053] Pig intestines were obtained from pig industry experimental station (MTT Agrifood Research Finland). The intestines were homogenized and plated on MRS as described in Example 1 .

Example 3. Characterization of bacterial colonies

[0054] Colonies selected from different samples and different cultivation conditions of Examples 1 and 2 were streaked as lines on fresh MRS- plates, and incubated at +30°C until the lines grew.

[0055] 15 ml of MRS medium was added into test tubes, each of which was then inoculated with a bacterial growth from a line on an MRS-plate. The tubes were incubated at +30°C until fully grown. The pH was then measured by using fresh MRS-medium as control. Isolates which gave rise to measurable lowering of pH (more than one unit) were chosen for further studies. The ability to lower the pH indicates ability to produce acid.

[0056] The potential CO2 production of the chosen isolates was determined with the Durham-tube test (Laborexin) according to the manufacturer's instructions. To this end, the chosen isolates were cultivated over night at +30°C in test tubes with 15 ml of MRS and the Durham-tube. The presence of absence of gas generated into the Durham-tube was determined. The pres- ence of gas indicates formation of CO ₂ and, consequently, heterofermentative type of carbohydrate utilization.