WO2012030268A1 - Antiinfective plant nucleoprotein isolates - Google Patents

Abstract

Foam fractionation is disclosed as a method for nucleoprotein capture and concentration from plant extracts without DNA or starch contamination. Plant nuclear proteins are first extracted by dilute acid or high-salt. Air is introduced as bubbles in the plant extract until foam formation. The foam is recovered and broken into a concentrated plant nuclear protein mixture which contains natural anti-infective histones, which are both antimicrobial and antiviral. The invention relates to a process for isolation of nucleoproteins from the plant kingdom, to the use thereof, or to a biocide and pharmaceutical product comprising an anti-infective nucleoprotein isolate. Plant histones prepared by foam capture represents an essentially unlimited source for natural, biodegradable peptide antibiotics and antivirals.

Description

ANTI-INFECTIVE PLANT NUCLEOPROTEIN ISOLATES

Global threats in the past, such as outbreak of flu and bioterrorism have further exemplified the need, and importance of hand hygiene in preventing the spread of infections. All these factors have contributed to increasing use of hand sanitizers in non-traditional settings such as schools, colleges, corporates, restaurants, fast food chains and food processing plants. There is a need for better household anti-infectives, wound care products, preventative feed or water additives and biodegradable sanitizers which do not pollute the environment, accumulate in the food chain or increasing the risk of antibiotics resistance, a significant problem in industrial-scale poultry fanning or during storage and transportation of fresh foods. Norovirus and Salmonella contamination are the leading causes of foodbome disease outbreaks, with poultry, beef, and leafy greens the most common foods involved. For example, if a chicken is contaminated with bacteria, washing it can spread bacteria on to work surfaces for up to a one-metre radius. A recent study by the UK Food Standards Agency found that 65% of raw shop-bought chicken is actually contaminated with the Campylobacter bug, which accounts for a third of all food-borne illnesses and commonly causes severe diarrhoea.

Campylobacter causes an estimated 30,000 cases of food poisoning a year in the UK only, including 15,000 hospitalisations and 80 deaths - more than E coli. Levels of the

controversial anti-bacterial additives triclosan and triclocarban present in the urine of people in the US jumped by over 40 per cent in two years.

Both triclosan and triclocarban can enter the food chain through the use of contaminated wastewater or fertilizer in agricultural fields.

Any eukaryotic cell nucleus contain equal amounts of DNA (deoxyribonucleic acid), histones and non-histones forming chromatin which is the physiologically relevant macromolecular complex of the genome. Histones are the cationic nucleoproteins with a very high proportion of positively charged amino acids arginine and lysine which package and compact the DNA of opposite negative charge in a 1 : 1 relationship. Histone modifications are also the backbone for epigenetic regulation and transcription. Animal histones were among the first proteins studied, in part because of their relative ease of isolation from bird erythrocyte nuclei. Apart being structural proteins, calf histones were reported to be broadspectrum anti-infectives by Hirsch J.G., J. Exp. Med 108, 925-944, 1958, through a mechanism which is not well understood. Moreover, some nonenveloped viruses such as adenovirus, norovirus and tobacco mosaic virus are also inhibited by histones (Ladygina M.E. et al., Vopr Virusol, 1978 Nov-Dec (6): 686-90; Tamura M. et al., Arch Virol, 148,1659-70, 2003). Noroviruses are estimated to cause 23 million cases of acute gastroenteritis (commonly called the "stomach flu") in the U.S. each year, and are the leading cause of gastroenteritis. Of viruses, only the common cold is reported more often than viral gastroenteritis (norovirus).

Histones are among the most highly conserved eukaryotic proteins, they are remarkably similar across the species, appearing as a family of five basic proteins with similar isoelectric points at pH 10.6 and with a molecular weight range 10 to 30 kDa: for example, there are only et al., Vopr Virusol, 1978 Nov-Dec (6): 686-90; Tamura M. et al., Arch Virol, 148,1659-70, 2003). Noroviruses are estimated to cause 23 million cases of acute gastroenteritis (commonly called the "stomach flu") in the U.S. each year, and are the leading cause of gastroenteritis. Of viruses, only the common cold is reported more often than viral gastroenteritis (norovirus).

Histones are among the most highly conserved eukaryotic proteins, they are remarkably similar across the species, appearing as a family of five basic proteins with similar isoelectric points at pH 10.6 and with a molecular weight range 10 to 30 kDa: for example, there are only two differences in the amino acid sequences of histone H4 in peas and cows. Such conservation suggests that these histones have crucial functions involving nearly all of their amino acids. Histones precipitate out of solution upon addition of sodium hydroxide to pH 10.6 and specifically by soy lecithin at pH 7.5 (Chargaff E. and Ziff M, J. Biol. Chem. 131, 25-34, 1939). Formation of salt-like compounds with phospholipids like lechitin in biological membranes could be the reason why histones at high concentration are toxic for mammalian endothelial cells during sepsis (Xu, J. et al., Nature Medicine 15, 1318-1321, 2009).

Plants and animals have developed antimicrobial (poly)peptides, which are able to form pores in the cytoplasmic membrane of invading microorganisms. Human skin and other epithelian linings are rich sources of peptide antibiotics, thus accomplishing an extraordinary resistance of these tissues to infection. Generally referred to as defensins, these peptides display the common feature of being both cationic and amphiphilic, exhibiting detergent-like activities via the direct biophysical disruption of bacterial cell membranes. Consequently, the mode of action is bacteriocidal rather than bacteriostatic, unlike the majority of existing antibiotics from molds which are targeting intracellular biochemical pathways. The direct lysis of the membranes minimizes the chances that bacterial resistance will arise. Accordingly, they imitate the early innate immunity response protecting the individual before any secondary, acquired immune response develops days or weeks post infection. Such a first line of defense is the immune protection system in many species including insects and amphibians, and remarkable well conserved during evolution. In the plant kingdom, correspondent defensin polypeptides are referred to as thionins whose activity are principally anti-fungal. The same defence concept probably exists in all multicellular organisms, and even down to the very bacteria themselves, where cationic peptides actually are used as competition agents for nutrition and survival (bacteriocins). All other antibacterial polypeptides described so far, are inducible and transcriptionally regulated. Learning from nature's defences against bacteria, cationic polypeptides are under

consideration for being a new classes of antibiotics and antivirals, provided that they could be made available in commercial quantities. This has not been accomplished so far from any biological material due to low recovery; alternative recombinant gene technology approaches are difficult due to the inherent cell permeating capabilities and toxicity of these substances in a fermentation environment.

Given the fact that histones are structurally almost identical between species, including all living plants, suggests an extraordinary shortcut possibility by direct extraction and use of anti-infective histones from the plant kingdom, according to EP 1420646. Animal and plant histones are completely interchangeable in the nucleosome core (Liberati-Langenbuch J. et al., BBRC 94, 1161-1168, 1980), and they are also inseparable from each other by any antimicrobial assay.

Europeean patent 1420646 issued to Rothman, teaches that plant histones which are extensively synthesized during plant cell division, are strong broadspectrum defensin-mimetic compounds whose biological activity are comparable with the tetracylines. They are unspecific just like all previous described bacteriocins, defensins or thionins. Moreover plant histones are effective against both gram(+)positive and gram(-)negative microbs, a rare feature among peptide antibiotics. Thus, plant histones perform several duties, besides being the DNA packaging material. Dissociated plant chromatin turns antibiotic ex vivo, due to extracted and liberated histones which can substitute or swap for natural endogenous histones while acting as a stand in for natural antibiotics across species.

The impact of EP 1420646 is that several of our most common edible plant commodities can be turned into peptide antibiotics, biocides, food preservatives or neutraceuticals. An advantageous antimicrobial histone extraction source for large-scale production are the common cereals as starting material because of their relatively large genome sizes, many exceeding 3,000 Mb/lC, corresponding to yields of 500 g histones from one hectare.

However, the complexity, instability and starch content of the initial extract makes the separation and isolation of nucleoproteins a complicated and economically demanding process. Modern protocols for histone extraction is based on solubility of histones in acids, either dilute hydrochloride acid or dilute 0.1 - 0.2M sulfuric acid which dissociate the nucleic acids from the histones but precipitates most other nuclear proteins and nucleic acids. Alternatively, high-salt extraction by 2M - 4M sodium chloride also dissociate chromatin at maintained neutral pH. The main downside to salt extraction is that cellular modifying enzymes and proteases can remain active for longer than in the acid-extraction method. It is therefore crucial that protease inhibitors are included during purification. According to the present invention protease control is obtained by the presence of sodium bisulphite and heat, > 55°C during the extractions.

Standard histone extraction protocols do not easily translate into plant use because of the inherently high starch content in plant parts causing stickiness and insolubility issues.

Moreover, plant histone preparations containing polyphenols also readily clouds or precipitate the separated proteins. During work with such procedures, however, it was noted that most preparations spontaneously foamed without agitation, which foam easily adhered to glass and plastic surfaces, usually considered an undesired behaviour in protein chemistry.

Astonishly, it was found that the very foam itself actually consists of the desired plant nucleoprotein mix of high plant histone content, leaving the DNA, polyphenols and starch behind in the original solution. The reason for this is that the cationic histone molecules behaves like natural detergents, even more so when they become denaturated and more hydrophobic during isolation.

Accordingly, the result of this unexpected observation is a most efficient process for isolating plant nucleoproteins which eliminates the starch problem at the same time.

Another result of the invention is that the inventive procedure is ideal for an automatic industrial extraction process that allows for an excellent and speedy isolation of a plant nucleoprotein of high histone content at low cost.

The present invention allows for a one-step plant nucleoprotein capture from solutions of low protein content without detergent addition, which is achieved by simply foaming acid or high- salt plant extracts with air, nitrogen or steam, recovering the rising anti-infective

nucleoprotein enriched foamate as the final product, which simultaneously become free of DNA and starch contamination, concentrated and desalted at the same time. Such surprising results suggest the possibility of extended use of a large number of plant food, animal feed preparations, vegetable oil extraction leftovers, co-products from the dry mill fuel or beverage ethanol process as starting material for anti-infective nucleoprotein production at large scale. Plant nucleoprotein foams can readily be prepared from dilute biomasses including those containing genome sizes of c-values less than 3,000 Mbp/lC, thus allowing an economical feasible extraction of the big four crops (conventional or GMOs), rapeseed (1,100 Mbp), soybean (1,100 Mbp), maize (2,500 Mbp), cottonseed (2,800 Mbp). Even smaller genomes from tomato (1,000 Mbp), apple (750 Mbp), grapevine (500 Mbp), common leftovers from the juice and wine industries, become realistic targets for extraction of nucleoprotein isolates by the present invention.

Historically, various flotation separation methods are widely used in wastewater treatment and mineral processing industries. Foam fractionation as a chemical purification method was first reported by Sebba (J Colloid interface Sci., 35 (4), 643, 1971) as 10-100 urn

microbubbles or colloidal gas aphrons created by intense stirring of a dilute surfactant in a solution resulting in clarification of suspensions. Proteins adsorb to to the bubble gas/liquid interface by hydrophobicity and electrostatics during concentration in the foam. Lalchew et al. (Biotechnology and Bioengineering, 24, 2253-2262, 1982) performed foam separation of DNA and proteins from laboratory mixtures of calf thymus chromatin in salt solutions. At a later stage, foam fractionation was applied for protein recovery by Jauregi et al. (Chemical Engineering Journal 65(1), 1-11,1997). Similarly, US patent No. 3,969,336, issued to

Criswell, teaches foam fractionation of whey proteins.

A preferred embodiment of the present invention is a continuous supply of compressed air fed through a plant extract, prepared by any chromatin dissociation method. Alternatively, a cruder plant histone preparation maybe obtained simply by placing the entire biomass in dilute acid containing protease-inhibiting sodium bisulphite during a prolonged period of time dissolving the nucleoproteins without an initial preparation of cell nuclei or chromatin, followed by steaming the whole batch at pH > 5.8, in a set-up otherwise similar to protein skimming in water purification which can be run automatically in a continuous online setup.

According to the present invention, plant histones do not require an additional detergent for foam formation because histones are inherently detergent-like molecules by themselves, already creating fairly stable foams upon shaking, thus allowing a self-supporting foam capture fractionation without addition of another detergent, in striking contrast to all known gas aphron methods which rely upon compulsory detergent addition for foam formation. According to the invention, the histones become denaturated and thermoresistant during isolation, which bring out foaminess obtained by bubbling without affecting their anti- infective properties.

A more comprehensive understanding of the invention can be obtained by considering the following Examples. However, it should be understood that the Examples are not intended to be unduly limitative of the invention.

Example 1

A 100 g sample of consumer wheat germs ( ungsornen, Sweden) were homogenized for 10 min at full speed in a Braun mixer in 400 ml tap water containing Triton X- 100 at 0.5% (w/v). An additional 100 ml of tap water were added, and the sample was stirred for 10 min. The debris was then removed by low-speed centrifugation (4000 xg for 5 min). The supernatant crude plant chromatin was aggregated by dropwise addition of 3M ammonium sulfate to a final concentration of 0.05 M. The chromatin was pelleted by centrifugation at 4000xg for 10 min. The supernatant was then decanted and the pellet resuspended in in 100 ml 0.1 x sodium citrate buffer + 5 mM sodium bisulfite. Such a 100 ml batch of crude plant chromatin was subject to dissociation into nucleic acid and chromosomal proteins by the addition of 25 ml 0.5M sulfuric acid to a final concentration of 0.1 M sulfuric acid prior to stirring for 6 hours. The dissociated chromatin material is still a complex mixture of DNA molecules, different histones and non-histones, further contaminated with starch as evidenced by strong blue reaction with iodine and resulting in turbidity and some insoluble components, hardly removable by any centrifugation attempt. The pH was adjusted to 7.0 with NaOH. Foam fractionation was performed in a 250 ml graduated glass cylinder fitted with an airstone (Aqua Fizzzz, USA) fed with humidified compressed air at about 0.90 cm/sek superficial gas velocity during 15 minutes at room temperature. The overflow was collected in 0.1% acetic acid. Freeze dried preparations are readily soluble in distilled water or saline. No starch was detected by iodine, but there was an intense reaction for Fast green which could be neutralized by heparin, indicating the presence of cationic protein which also forms a precipitation by 0.1% soy lecithin at pH 7.5, a ubiquitous feature of the histones. Such insoluble salt-like complexes with phospholipids are split by dimethylether, 95% ethanol at 60°C or 2- pentanone. The tests are indicative of the presence of histones in the nucleoprotein mix that form foam. The anti-infective activity of the foam fractionated cationic nucleoprotein preparation was successfully tested in the Hygicult® (Orion Diagnostica Oy, Finland) system for hygiene control in a kitchen environment harbouring both gram-positive and gram-negative bacteria.

Example 2

A cruder nucleoprotein preparation is obtained in a most simple way by directly and simultainously contacting the biomass starting material with dilute acid without the initial chromatin purification step according to Example 1. Thus, 100 g raw wheat germs were incubated overnight in room temperature on rotator in 500 ml 0.4M sulfuric acid, then adjusted with NaOH to pH 6.0 before foam fractionation as in Example 1. The foam exhibit distinct anti-infective properties in Hygicult® assays.

Example 3

Wheat germs (100 g) was suspended in 4M NaCI in the cold overnight and homogenized for about 10 minutes in a Braun mixer. The high salt concentration dissociate the plant chromatin into DNA and proteins. The homogenate was stepwise diluted with distilled water to 0.3M NaCl, filtered through a nylon sieve of 0.3 mm, and the filtrate (at 0.3M NaCl) was subjected to the foam fractionation process at pH 6.0 at similar parameters as in Example 1. The collected foamate is equally antimicrobial.

Example 4

ExPro rapeseed meal (200 g, AarhusKarlshamn, Sweden), a byproduct following rape seed oil extraction, was homogenized during 4 hours in 2,000 ml 4M NaCl and further diluted to 0.3M before the plant material was contacted with steam. This was performed by directing a 19 bar pressurized water vapor jet from an espresso/coffee machine (Jura Impressa 501, Switzerland) towards the homogenate surface until frothing. The overflow foam was allowed to settle in 0.1% acetic acid before freezing at -20°C. The product is an easily soluble plant histone mixture, stripped from contaminating DNA and starch, exhibiting a strong antimicrobial activity in the Hygicult® test system.

Example 5

Seventy liters of distillers dried grains with solubles (DDGS), a wet byproduct (water content 90%; 416g protein/kg dried state; original pH 3.9) following ethanol fermentation (Nobbelov, Sweden), was added solid sodium chloride to 0.3 M and steamed at approximately pH 6.0 for

Claims

Claims:

1. A process for simultaneous removal of carbohydrates, DNA and polyphenols from biomasses irrespectively of nuclear genome size, simultaneously isolating, concentrating and desalting plant cationic nucleoproteins with anti-infective properties, comprising the steps of: aqueous plant chromatins are prepared by conventional purification procedures such as ammonium salt aggregation;

said plant chromatins are further extracted for nucleoprotein by acid extraction or high-salt extraction;

subjecting said plant dissociated plant chromatin to a foam capturing system without detergents;

by entrainment of gas bubbles of air, nitrogen or hot steam through said plant chromatin extract during foam formation allowing cationic nucleoproteins to accumulate in the rising foam on top of the reaction vessel, leaving the DNA and starch behind;

said foam is collected and allowed to collapse yielding a foamate enriched with said cationic nucleoproteins.

2. The process of claim 1, wherein said biomass is selected from the group consisting of animal green fodder such as alfalfa, barley, birdsfoot trefoil, brassicas, cassava, clover, cottonseed, grasses including rice, jatropha, maize, millet, oats, palm kernel cake, rapeseed, sorghum, soybeans, sunflower, wheat and its byproducts obtained by milling for flour, vegetable oil press cake, seeds, grains and germs either whole or prepared by crushing or milling, sprouted grains, legumes, seaweed, marine macro-algae, micro-algae in ponds or bioreactors, yeasts and combinations or blends thereof.

3. The process of claim 1, wherein said biomass starting material is maize and cereal distillers grains which are co-products of bioethanol production.

4. The process of claim 1, wherein said plant chromatin has desoxyribonucleic acid (DNA) c- value less than 3,000 megabase pairs (Mbp).

5. The process of claim 1, wherein said foam is generated by a continuously frothing water steam jet positioned at the top boundary of the solution containing said plant chromatin preparation while maintaining the temperature of the liquid system from about +55°C to about +70°C, creating a plant nucleoprotein enriched foamate.

6. A process for separating cationic nucleoproteins by simply employing foam capturing on a biomass independent of nuclear genome size, following extracting the entire plant material in dilute mineral acid and disregarding any initial chromatin isolation procedure, comprising the steps of:

an aqueous biomass slurry is prepared;

such biomass slurry is contacted with dilute acid in order to dissolve cationic nucleoproteins from such biomass material; the pH of the preparation is raised to pH 5.8 - pH 10.0;

introducing said acid extracted biomass preparation to a foam fractionator operating above pH 6.0;

the foam fractionator is fed with gas bubbles or steam through said extract, wherein cationic nucleoproteins preferentially adsorb to a gas-liquid interface of the bubbles during formation of a foam at the top of the reaction vessel;

said foam collapses into a foamate enriched with the desired cationic nucleoproteins.

7. The process of claim 6, wherein said biomass is selected from the group consisting of animal green fodder such as alfalfa, barley, birdsfoot trefoil, brassicas, cassava, clover, cottonseed, grasses including rice, jatropha, maize, millet, oats, palm kernel cake, rapeseed, sorghum, soybeans, sunflower, wheat and its byproducts obtained by milling for flour, vegetable oil press cake, seeds, grains and germs either whole or prepared by crushing or milling, sprouted grains, legumes, seaweed, marine macro-algae, micro-algae in ponds or bioreactors, yeasts and combinations or blends thereof.

8. The process of claim 6, wherein said biomass starting material is maize and cereal distillers grains which are co-products of bioethanol production.

9. The process of claim 6, wherein said biomass starting material contain desoxyribonucleic acid (DNA) with a c-value less than 3,000 megabase pairs (Mbp).

10. The process of claim 6, wherein said foam is generated by a continuously frothing water steam jet positioned at the top boundary of the solution containing said plant preparation while maintaining the temperature of the liquid system from about +55°C to about +70°C, creating a plant nucleoprotein enriched foamate.

such biomass slurry is contacted with dilute acid in order to dissolve cationic nucleoproteins from such biomass material;

the pH of the preparation is raised to pH 5.8 - pH 10.0;

introducing said acid extracted biomass preparation to a foam fractionator operating above pH 6.0;

the foam fractionator is fed with gas bubbles or steam through said extract, wherein cationic nucleoproteins preferentially adsorb to a gas-liquid interface of the bubbles during formation of a foam at the top of the reaction vessel;

said foam collapses into a foamate enriched with the desired cationic nucleoproteins.

7. The process of claim 6, wherein said biomass is selected from the group consisting of animal green fodder such as alfalfa, barley, birdsfoot trefoil, brassicas, cassava, clover, cottonseed, grasses including rice, jatropha, maize, millet, oats, palm kernel cake, rapeseed, sorghum, soybeans, sunflower, wheat and its byproducts obtained by milling for flour, vegetable oil press cake, seeds, grains and germs either whole or prepared by crushing or milling, sprouted grains, legumes, seaweed, marine macro-algae, micro-algae in ponds or bioreactors, yeasts and combinations or blends thereof.

8. The process of claim 6, wherein said biomass starting material is maize and cereal distillers grains which are co-products of bioethanol production.

9. The process of claim 6, wherein said biomass starting material contain desoxyribonucleic acid (DNA) with a c-value less than 3,000 megabase pairs (Mbp).

10. The process of claim 6, wherein said foam is generated by a continuously frothing water steam jet positioned at the top boundary of the solution containing said plant preparation while maintaining the temperature of the liquid system from about +55°C to about +70°C, creating a plant nucleoprotein enriched foamate.