Macroalgal fermentation for the production of vitamin k2

Abstract

The present invention relates to a fermentation process utilizing seaweed biomass co-inoculated with a yeast strain and a carbohydrate source, wherein the fermentation process is performed in the presence of one or more Bacillus strains to increase the production of vitamin K2. In one embodiment, the seaweed is Kappaphycus alvarezii, the yeast is Saccharomyces cerevisiae, Candida utilis, or the mixture thereof, and the one or more Bacillus strains is Bacillus altitudinis, Bacillus pumilus, and / or Bacillus subtilis.

Description

Macroalgal fermentation for the production of vitamin K2

[0001] The present invention relates to a fermentation process to produce vitamin K2 from macroalgal biomass.BACKGROUND

[0002] Fermentation, a natural process extensively utilized to enhance the quality of raw materials, significantly improves palatability, nutrient content, and the presence of bioactive constituents in various substrates. Seaweed benefits substantially from fermentation, exhibiting remarkable potential as a food and feed additive, ingredient, supplement, or meal. This process can be specifically tailored to endow the seaweed biomass with particular nutritional and health-promoting properties, catering to human and targeted animal needs.

[0003] Vitamin K2, also known as menaquinone, represents one of the several forms of fat-soluble vitamin K, crucial for blood clotting, bone health, and other physiological functions. Distinct from vitamin K1 (phylloquinone), primarily associated with blood clotting, vitamin K2 predominantly influences bone metabolism and cardiovascular health. A notable source of vitamin K2 is fermented products, with natto (fermented soy meal) being a prime example.

[0004] Kappaphycus alvarezii, a species within the Kappaphycus genus, is a highly versatile macroalgae, known for its diverse applications. Said seaweed harbors an ecosystem of endemic bacteria, predominantly of the Bacillus species. These bacteria play an important role in the seaweed's ecological and physiological processes. The presence of Bacillus spp. in Kappaphycus alvarezii not only contributes to the alga's health and growth but also influences its chemical composition. This symbiotic relationship enhances the macroalgae's utility in various applications, ranging from agricultural biofertilizers to potential sources of bioactive compounds in the pharmaceutical sector. The rich composition of Kappaphycus alvarezii, intertwined with the biological activities of Bacillus spp., underpins its utility in industries such as food, cosmetics, and biotechnology, making it a focus of increasing interest in the scientific and commercial sectors.

[0005] US 2023 / 0235375 discloses a sustainable fermentation process designed for increased efficiency and reduced environmental impact. This process discloses a production method and extraction of two or more fermentation products in a single fermentation cycle. Specifically, it can produce a "primary" and a "secondary" fermentation product, where one is a water-soluble organic compound and the other a fat-soluble organic compound, preferably a vitamin K2. The fermentation process includes cultivating a host cell, in particular a genetically modified host cell, under suitable conditions forproducing said fermentation products. At the end of the process, the products are extracted or isolated from the biomass, which is discarded or treated to eliminate any harmful recombinant DNA.

[0006] US 2011 / 0250635 discloses a method and system for processing biomass, utilizing an apparatus with an innovative housing and rotor design. The process comprises placing biomass in a fluid medium and inducing cavitation to partially separate it. The biomass and fluid are then sent into the housing, where the rotor, equipped with a variety of protrusions, further separates the biomass. Additionally, the method includes heating the biomass and the fluid wherein the said fluid's acidity is in a pH range between 2 to 6. The system's rotors feature distinct designs, such as abutting, alternating height protrusions, grooves, and rows of protrusions spaced no less than approximately 6 mm apart.

[0007] US 2022 / 0273571A1 discloses compositions and methods related to probiotics. The invention involves probiotics encapsulated in a matrix of protein and carbohydrates derived from non-fermented biomass, wherein said encapsulation enhances the probiotics' viability. Said compositions are used in methods for promoting health in a subject, particularly targeting the gut microbiome. The encapsulation process comprises mixing the probiotics with protein and carbohydrates from the biomass to form a powder. This approach aims to address rising chronic diseases by leveraging the beneficial effects of probiotics on gut health, offering a preventative and clinical solution to global health challenges.

[0008] US 2012 / 0137574A1 describes systems and methods for harvesting microalgae using zooplankton. This process includes feeding microalgae to zooplankton, gathering the zooplankton, extracting lipids from them, and processing these lipids into biofuels. Optionally, zooplankton can also be fed to zooplanktivorous fish, with subsequent lipid extraction and processing. These systems and methods are applicable in open outdoor algae culturing facilities and closed systems comprising photobioreactors. This invention describes several advantages such as increased microalgal biomass recovery, additional food sources for certain fish, direct lipid extraction from zooplankton, and stabilization of plankton community structure.

[0009] The examples described herein are not limited to examples which solve problems mentioned in this background section.SUMMARY OF THE INVENTION

[0010] Examples of preferred aspects and embodiments of the invention are as set out in the accompanying independent and dependent claims.

[0011] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intendedto identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0012] A first aspect the invention relates to a method for obtaining one or more products with a content of vitamin K2, the method comprising:(a) inoculating an algae biomass with a yeast strain and a carbohydrate source; and(b) performing a fermentation process on the algae biomass inoculated with a yeast strain and a carbohydrate source, wherein the fermentation process is performed in the presence of one or more Bacillus strains to increase the production of vitamin K2; wherein the one or more Bacillus strains are naturally occurring on the algae or isolated cultures.

[0013] In some embodiments, the method further comprises performing an enzymatic treatment of the algae biomass material at a temperature between 30°C and 40°C.

[0014] In some embodiments, the method further comprises inoculating the enzymatically treated algae biomass with at least one yeast strain in the presence of a carbohydrate source.

[0015] In some embodiments, the method further comprises performing a fermentation process on the algae biomass at a pH between 6.5 and 4.0.

[0016] In some embodiments, the method further comprises recovering one or more fermentation products from the fermentation process.

[0017] In some embodiments, the one or more Bacillus strains are selected from the group consisting of Bacillus pumilus, Bacillus altitudinis, and Bacillus subtilis.

[0018] In some embodiments, the products have a higher content of vitamin K2 than a product or products obtained from a method comprising a fermentation process performed on an algae biomass in the absence of a yeast strain and one or more Bacillus strains.

[0019] In some embodiments the one or more products are 1 , 2 or 3 products.

[0020] In some embodiments, the one or more products are the same product.

[0021] In some embodiments, the one or more products are 2 products.

[0022] In some embodiments, the one or more products are 3 products.

[0023] In some embodiments, each of the one or more products are independently in the form of liquid extract, fermented solid biomass, or dried fermented biomass.

[0024] In some embodiments, the one or more products are in the form of liquid extract.

[0025] In some embodiments, the one or more products are in the form of dried fermented biomass.

[0026] In some embodiments, the one or more products are in the form of fermented solid biomass.

[0027] In some embodiments, the algae material is a seaweed material.

[0028] In some embodiments, the algae biomass is selected from the group consisting ofKappaphycus spp, Ascophyllum nodosum, Fucus vesiculosus, Laminaria japonica, Ulvalactuca, Sargassum spp., Porphyra spp., Eucheuma spp., Macrocystis pyrifera (commonly known as giant kelp), Gracilaria spp.

[0029] In some embodiments, the algae biomass is Kappaphycus alvarezii.

[0030] In some embodiments, the one or more Bacillus strains are isolated from algae material.

[0031] In some embodiments, the method further comprises isolating the one or more Bacillus strains from algae material.

[0032] In some embodiments, the isolating the one or more Bacillus strains from algae material comprises:(a) collecting algae material from different sites and placing them in sterile containers;(b) treating the algae material with ethanol, followed by rinsing with sterile seawater;(c) homogenizing and cut in small piece the algae material, in sterile seawater;(d) performing serial dilutions of the homogenate and plating aliquots onto marine agar to culture bacteria;(e) incubating the plates to allow bacterial colonies to grow;(f) selecting and purifying individual colonies to obtain pure Bacillus strains.

[0033] In some embodiments there is provided a method for producing vitamin K2, comprising: a) implementing a method as provided herein; and b) recovering the vitamin K2.

[0034] In some embodiments, the fermentation process is performed in a fermentation tank.

[0035] In some embodiments, the liquid extract exhibits a vitamin K2 concentration ranging from 1 ,000 to 1,100 micrograms per 100 grams.

[0036] In some embodiments, the dried biomass exhibits a vitamin K2 concentration ranging from 100 to 150 micrograms per 100 grams.

[0037] In some embodiments, the fermented seaweed solid biomass in its wet form exhibits a vitamin K2 concentration ranging from 2,000 to 3,000 micrograms per 100 grams.

[0038] In some embodiments, the one or more Bacillus strains is a pure culture of Bacillus.

[0039] In some embodiments, the one or more Bacillus strains comprises a mixture of Bacillus strains.

[0040] In some embodiments, the one or more Bacillus strains are one or more endemic Bacillus strains.

[0041] In some embodiments, the one or more Bacillus strains increase the production of vitamin K2 content in the one or more products compared to the vitamin K2 content produced in one or more products obtained from a method comprising a fermentation process performed on an algae biomass in the absence of the one or more Bacillus strains.

[0042] In some embodiments, wherein the one or more products is liquid extract, the increased vitamin K2 content is 1 ,000 to 1 ,100 micrograms per 100 grams.

[0043] In some embodiments, wherein the one or more products is dried biomass, the increased vitamin K2 content is 100 to 150 micrograms per 100 grams.

[0044] In some embodiments, wherein the one or more products is fermented seaweed solid biomass in its wet form, the increased vitamin K2 content is 2,000 to 3,000 micrograms per 100 grams.

[0045] A second aspect of the invention relates to a method for increasing the quantity of vitamin K2 obtained by fermenting algae biomass, the method comprising:(a) performing an enzymatic treatment of the algae biomass material at a temperature between 30°C and 40°C;(b) inoculating the enzymatically treated algae biomass with at least one yeast strain in the presence of a carbohydrate source;(c) performing a fermentation process on the algae biomass at a pH between 6.5 and 4.0;(d) recovering one or more fermentation products from the fermentation process; wherein the fermentation process is performed in the presence of one or more Bacillus strains capable of producing and / or enhancing the production of vitamin K2, wherein said one or more Bacillus strains are naturally present on the algae or isolated cultures.

[0046] In some embodiments, the method further comprises performing an enzymatic treatment of the algae biomass material at a temperature between 30°C and 40°C.

[0047] In some embodiments, the method further comprises inoculating the enzymatically treated algae biomass with at least one yeast strain in the presence of a carbohydrate source.

[0048] In some embodiments, the method further comprises performing a fermentation process on the algae biomass at a pH between 6.5 and 4.0.

[0049] In some embodiments, the method further comprises recovering one or more fermentation products from the fermentation process.

[0050] In some embodiments, the one or more Bacillus strains are selected from the group consisting of Bacillus pumilus, Bacillus altitudinis, and Bacillus subtilis.

[0051] In some embodiments, the products have a higher content of vitamin K2 than a product or products obtained from a method comprising a fermentation process performed on an algae biomass in the absence of a yeast strain and one or more Bacillus strains.

[0052] In some embodiments the one or more products is 1 , 2 or 3 different products.

[0053] In some embodiments, the one or more products are the same product.

[0054] In some embodiments, the one or more products are 2 products.

[0055] In some embodiments, the one or more products are 3 products.

[0056] In some embodiments, each of the one or more products are independently in the form of liquid extract, fermented solid biomass, or dried fermented biomass.

[0057] In some embodiments, the one or more products are in the form of liquid extract.

[0058] In some embodiments, the one or more products are in the form of dried fermented biomass.

[0059] In some embodiments, the one or more products are in the form of fermented solid biomass.

[0060] In some embodiments, the algae material is a seaweed material.

[0061] In some embodiments, the algae biomass is selected from the group consisting ofKappaphycus spp, Ascophyllum nodosum, Fucus vesiculosus, Laminaria japonica, (Jiva iactuca, Sargassum spp., Porphyra spp., Eucheuma spp., Macrocystis pyrifera (commonly known as giant kelp), Gracilaria spp.

[0062] In some embodiments, the algae biomass is Kappaphycus alvarezii.

[0063] In some embodiments, the one or more Bacillus strains are isolated from algae material.

[0064] In some embodiments, the method further comprises isolating the one or more Bacillus strains from algae material.

[0065] In some embodiments, the isolating the one or more Bacillus strains from algae material comprises:(a) collecting algae material from different sites and placing them in sterile containers;(b) treating the algae material with ethanol, followed by rinsing with sterile seawater;(c) homogenizing and cut in small piece the algae material, in sterile seawater;(d) performing serial dilutions of the homogenate and plating aliquots onto marine agar to culture bacteria;(e) incubating the plates to allow bacterial colonies to grow;(f) selecting and purifying individual colonies to obtain pure Bacillus strains.

[0066] In some embodiments there is provided a method for producing vitamin K2, comprising: a) implementing a method as provided herein; and b) recovering the vitamin K2.

[0067] In some embodiments, the fermentation process is performed in a fermentation tank.

[0068] In some embodiments, the liquid extract exhibits a vitamin K2 concentration ranging from 1 ,000 to 1,100 micrograms per 100 grams.

[0069] In some embodiments, the dried biomass exhibits a vitamin K2 concentration ranging from 100 to 150 micrograms per 100 grams.

[0070] In some embodiments, the fermented seaweed solid biomass in its wet form exhibits a vitamin K2 concentration ranging from 2,000 to 3,000 micrograms per 100 grams.

[0071] In some embodiments, the one or more Bacillus strains is a pure culture of Bacillus.

[0072] In some embodiments, the one or more Bacillus strains comprises a mixture of Bacillus strains.

[0073] In some embodiments, the one or more Bacillus strains are one or more endemic Bacillus strains.

[0074] In some embodiments, the one or more Bacillus strains increase the production of vitamin K2 content in the one or more products compared to the vitamin K2 content produced in one or more products obtained from any one of the methods as described herein comprising a fermentation process performed on an algae biomass in the absence of the one or more Bacillus strains.

[0075] In some embodiments, wherein the one or more products is liquid extract, the increased vitamin K2 content is 1 ,000 to 1 ,100 micrograms per 100 grams.

[0076] In some embodiments, wherein the one or more products is dried biomass, the increased vitamin K2 content is 100 to 150 micrograms per 100 grams.

[0077] In some embodiments, wherein the one or more products is fermented seaweed solid biomass in its wet form, the increased vitamin K2 content is 2,000 to 3,000 micrograms per 100 grams.

[0078] A further aspect of the invention relates to use of one or more isolated Bacillus strains for obtaining vitamin K2 from algae biomass.

[0079] In some embodiments the one or more isolated Bacillus strains is a pure Bacillus strain.

[0080] In some embodiments, the one or more Bacillus strains comprises a mixture of Bacillus strains.

[0081] It will be apparent to anyone of ordinary skill in the art, that some of the features indicated in relation to the methods of the present disclosure will also be preferred aspects of the uses as disclosed herein.

[0082] It will also be apparent to anyone of ordinary skill in the art, that some of the preferred features indicated above as preferable in the context of one of the aspects of the disclosed technology indicated may replace one or more preferred features of other ones of the preferred aspects of the disclosed technology. Such apparent combinations are not explicitly listed above under each such possible additional aspect for the sake of conciseness.

[0083] Other examples will become apparent from the following detailed description, which, when taken in conjunction with the drawings, illustrate by way of example the principles of the disclosed technology.BRIEF DESCRIPTION OF THE DRAWINGS

[0084] FIG 1. shows the process step of fermented seaweed to improve vitamin K2 content.

[0085] FIG. 2 shows the microbial 16s rRNA sequence of endemic bacteria isolated from seaweed with the capacity of producing Vitamin K2 and their identification.

[0086] The accompanying drawings illustrate various examples. The skilled person will appreciate that the illustrated element boundaries (e.g., boxes, groups of boxes, or other shapes) in the drawings represent one example of the boundaries. It may be that in some examples, one element may be designed as multiple elements or that multiple elements may be designed as one element. Common reference numerals are used throughout the figures, where appropriate, to indicate similar features.DETAILED DESCRIPTION

[0087] The following description is made for the purpose of illustrating the general principles of the present technology and is not meant to limit the inventive concepts claimed herein. As will be apparent to anyone of ordinary skill in the art, one or more or all of the particular features described herein in the context of one embodiment are also present in some other embodiment(s) and / or can be used in combination with other described features in various possible combinations and permutations in some other embodiment(s).

[0088] In the following, exemplary embodiments of the invention will be described, referring to the figures. These examples are provided to provide further understanding of the invention, without limiting its scope.

[0089] In the following description, a series of steps are described. The skilled person will appreciate that unless required by the context, the order of steps is not critical for the resulting configuration and its effect. Further, it will be apparent to the skilled person that irrespective of the order of steps, the presence or absence of time delay between steps, can be present between some or all of the described steps.

[0090] The process and material according to the invention are illustrated in the nonlimiting embodiments that follow.

[0091] In general, the present invention relates to a fermentation process of algae biomass for the production of vitamin K2. In one embodiment, the present invention provides a process wherein the fermentation of the algae biomass is performed in a semisolid state and under aerobic conditions. In one embodiment, a product of the fermentation process of algae biomass comprises a biomass substrate containing a high vitamin K2 content.

[0092] In one embodiment, the fermentation process comprises the steps of: coinoculated the algae biomass with yeast starter cultures. This embodiment is characterized by a synergistic interaction between the algae biomass and the added yeast, increasing the vitamin K2 content of the fermentation products. In one embodiment, the present invention relates to a fermentation process that employs algae biomass, particularly seaweed, as the fermentation substrate. Suitable seaweed species for thispurpose include, but are not limited to: Kappaphycus spp, Ascophyllum nodosum, Fucus vesiculosus, Laminaria japonica, Ulva lactuca, Sargassum spp., Porphyra spp., Eucheuma spp., Macrocystis pyrifera (commonly known as giant kelp), Gracilaria spp, or other macroalgae and microalgae species.

[0093] In a preferred embodiment, the fermentation process utilizes a seaweed biomass comprising at least one seaweed selected from the group consisting of Kappaphycus alvarezii, Kappaphycus spp, or other Kappaphycus species. Additionally, another embodiment of the invention utilizes yeast species such as Saccharomyces cerevisiae, Candida utilis, among others, in the fermentation process.

[0094] In one embodiment, the biomass of macroalgae and / or microalgae utilized in the fermentation process contains a mixture of bacteria strains, including but not limited to Bacillus pumilus, Bacillus altitudinis, and Bacillus subtilis. These bacteria strains are naturally present on the macroalgae and / or microalgae and are capable of producing vitamin K2 during fermentation. The presence of these endogenous bacteria enhances the vitamin K2 synthesis within the biomass, contributing to the overall efficiency and efficacy of the fermentation process disclosed in this invention.

[0095] In one particular embodiment, the isolation process of the bacilli from seaweed comprises several steps, including but not limited to: a) Seaweed samples are collected from different farm sites and placed in sterile containers to minimize the possibility of contamination. After, the samples are rinsed in sterile seawater or phosphate-buffered saline (PBS) to remove loosely attached microbes and debris.(b) The Seaweed samples are dipped in ethanol for a duration ranging between 30 to 60 seconds. After the ethanol treatment the seaweed samples are thoroughly rinsed with sterile seawater. This helps focus on endophytic or tightly attached bacteria by eliminating surface contaminants.(c) Two to three grams of seaweed are cut into small pieces using sterile scissors. The cut samples are homogenized in 10 mL of sterile seawater using a sterile mortar and pestle or a stomacher.(d) Serial dilutions are performed by diluting the homogenate from 10-1to 10“6using sterile seawater or PBS. An aliquot from each dilution is plated onto marine agar plates to culture the bacteria.(e) The plates are incubated at 25-30°C for 3-7 days, and colony growth is monitored daily.(f) Individual colonies with distinct morphologies are selected. The selected colonies are re-streaked onto fresh marine agar plates to obtain pure isolates, wherein the Isolates are pure cultures derived from heterogeneous wild-type microbial populations. At least 2 to 3 rounds of streaking are performed to ensure purity.(g) Gram staining is used to determine the approximate classification of the bacterial species of the isolates and identify bacillus types. Catalase, oxidase, and other relevant biochemical assays are performed to further characterize the bacterial strains.(h) Genomic DNA is extracted from the purified bacterial isolates After, the 16S rRNA gene is amplified with a polymerase chain reaction (PCR) with universal bacterial primers. The amplified 16S rRNA gene sequences are analyzed and compared with sequences in databases such as NCBI GenBank for species identification.

[0096] The bacilli isolated through this method have demonstrated the capabilities of producing vitamin K2 during the seaweed fermentation process. Due to this peculiarity, these Bacillus species — including but not limited to Bacillus pumilus, Bacillus altitudinis, and Bacillus subtilis — have demonstrated the capabilities to synthesize vitamin K2 during fermentation processes and enhances the vitamin content of the final fermented product. The bacilli strains isolated using the described method can be utilized in any fermentation protocol suitable for the production of vitamin K2. By incorporating these specific strains into suitable fermentation protocols, the production of vitamin K2 can be significantly increased.

[0097] In a specific embodiment the use of Kappaphycus spp. as a substrate combined with a step of an inoculation of Saccharomyces cerevisiae in the fermentation process, significantly boosts the vitamin K2 yield through a synergistic effect. As a result of this synergy, in a specific embodiment the fermentation process disclosed in this invention comprises a self-fermentation mechanism with the addition of yeast inoculation show a high production rate of vitamin K2 in the seaweed biomass.

[0098] In one embodiment, the fermentation process of seaweed biomass for the production of vitamin K2 according to the present invention comprises several steps, including but not limited to:(a) Washing and shredding the seaweed raw material;(b) perform an enzymatic treatment of the seaweed raw material at a temperature comprising between 30°C and 40°C;(c) inoculating the seaweed raw material with yeast and a carbohydrate source;(d) carry out the fermentation process at a pH of 6.5 to 4.0 and finish the fermentation process when the pH reaches a value between 3.5 and 4.5; and(e) after completing the fermentation process a recovery and / or extraction of the fermentation products can be performed.

[0099] In accordance with one embodiment of the present invention, the fermentation process provides one or more fermentation products in solid and / or liquid form. In one particular embodiment, said products contain an improved vitamin K2 content and can be in the form of extracted liquid, fermented solid biomass, or dried fermented biomass.

[0100] Referring now to FIG. 1, a flow chart illustrating the seaweed biomass process in accordance with an embodiment of the invention is shown. This embodiment of the seaweed biomass fermentation process encompasses several distinct steps. Initially, it involves a pre-treatment preparation stage for the seaweed, followed by the preparation of the yeast culture. Subsequently, the seaweed biomass is subjected to a mixing step according to the invention in the presence of yeast, and the obtained biomass is transferred into a fermentation tank. At the end of the fermentation process, the recovery and / or extraction of the fermentation product is performed.

[0101] In one embodiment, the pre-treatment preparation of seaweed raw material comprises the following steps:(a) The seaweed pre-treatment preparation process includes a receiving and storage step for receiving and storing the seaweed raw material in optimal conditions;(b) a cleaning step for washing said seaweed raw material;(c) and subsequently, the process incorporates a step for blanching said seaweed raw material;(d) a mechanical treatment is employed to reduce the seaweed raw material into smaller particles.

[0102] In one embodiment, the seaweed raw material used in the fermentation process according to the present invention comprises semi-dried seaweed and / or fresh seaweed. Additionally, in another embodiment the cleaning step comprises soaking of the seaweed raw material in fresh water for a duration between 2 and 5 hours, as a result of this treatment, contaminants and debris are removed from the seaweed raw material.

[0103] In one embodiment, the yeast culture preparation process includes a receiving and storage step for receiving and storing the yeast in optimal conditions and a measurement step to measure the amount of yeast required in the fermentation process. In one embodiment said fermentation inoculant is prepared at a minimum of 6 hours earlier and not more than 32 hours before mixing said inoculant with the seaweed raw material. In one embodiment the composition of the fermentation inoculant includes a mixture of yeast, sugar, enzyme, and water. In this embodiment the sugar is selected from the group consisting of sucrose or table sugar. In this embodiment the enzyme is a commercial cellulase enzyme. In one particular embodiment, said enzyme is derived from Trichoderma reesei. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Candida utilis. In some embodiments the yeast is Saccharomyces cerevisiae. In some embodiments the yeast is Candida utilis. In some embodiments the yeast is Saccharomyces cerevisiae and Candida utilis at a ratio of 1:0.2- 1.

[0104] In accordance with one embodiment of the present invention, the semi-dried seaweed material is treated with a blanching process in hot water at a temperature ofmore than 85°C and for a duration between 15 and 35 minutes. As a result, the said blanching process eliminates surface microbials, silt, mud, and other contaminant from the semi-dried seaweed. In another alternative embodiment, the blanching stage is not necessary for the fresh seaweed material.

[0105] In a specific embodiment of the present invention, the mechanical pretreatment stage of the seaweed raw material used in the fermentation process disclosed in this invention includes a grinding phase, which is executed using a meat grinder or another appropriate cutting tool. As a result, the size of the seaweed raw material is reduced to a range between 2 and 8 mm. In some embodiments the size of the seaweed raw material is reduced to a range between 3 and 8 mm.

[0106] In one embodiment of the present invention, the fermentation process starts with a mixing step of yeast culture with the seaweed raw material to form a mixture. During this mixing step, a quantity of filtered water is added to the mixture to ensure that said mixture reaches a consistency that can be easily agitated by a paddle. In another embodiment, an alternative agitation method is employed, wherein the mixture is agitated by a pulsating air. In another embodiment, in the mixing step the fermentation ingredient is mixed using a hand mixer until the mixture is homogenized. In this embodiment, the mixing step typically is completed within a timeframe ranging from 3 to 10 minutes.

[0107] Following these initial stages, the fermentation process continues with additional steps to further optimize vitamin K2 production.

[0108] In another embodiment, following one or more of the preceding treatments, the resulting ad ixture is transferred into a fermentation tank, where it serves as the biomass for fermentation. In one embodiment of the process of the invention, one or more mixing and / or agitation steps are performed in the fermentation tank during the fermentation process. In one embodiment said mixing and / or agitation steps are performed at a frequency of at least one every 24 hours. In this embodiment, the fermentation process in the tank is performed at a preferred temperature in the range between 29°C to 35°C. In one embodiment, the temperature measures in the fermentation tank are taken through a probe inserted 10 centimetres onto the surface of the biomass to measure the surface temperature of said biomass and to measure the temperature in the centre region of the biomass. In a specific embodiment, a 20 centimetres wide hole with about 60 centimetres depth was dug into the biomass to obtain the temperature. In a preferred embodiment, the average temperature is calculated from two or more temperature measurements, wherein said measurements are performed at predetermined time intervals.

[0109] In another embodiment, the pH of the biomass within the fermentation tank is monitored every 12 hours. The fermentation process is completed when the pH reaches a value between 3.5 and 4.5. To measure the pH of the biomass, the following steps is employed:(a) A hole approximately 20 cm wide and 60 cm deep is excavated into the biomass.(b) A 50 mL sample of the liquid accumulating in the hole is collected using a cup.(c) The pH value of the sampled liquid is measured using a portable pH meter.

[0110] In one embodiment, after the completion of the fermentation process, the liquid component is separated from the fermented biomass using methods such as straining or directly draining from the fermentation tank. Subsequently, the solid biomass is transferred to a drying facility. In another embodiment, the extracted liquid is collected and dehydrated using a spray dryer or a freeze dryer, resulting in a concentrated product. In another embodiment, the dried fermented biomass is processed through a grinding step and subsequently proceeds to the packaging step, where the material is prepared for storage and distribution.

[0111] In one embodiment, the fermented products of the disclosed invention contain an improved vitamin K2 content, and said products are available in various forms, including: extracted liquid: a liquid with a high content of vitamin K2, obtained from the fermentation process; fermented Solid Biomass: a solid biomass resulting from fermentation, with a high content of vitamin K2; or dried fermented biomass: a fermented biomass that has been dried, preserving its high vitamin K2 content for extended shelf life and ease of handling.

[0112] In one particular embodiment, the liquid extract show a vitamin K2 concentration ranges from 1000 to 1100 micrograms per 100 grams. In another embodiment, the fermented solid biomass in its wet biomass form, show a vitamin K2 concentration ranges from 2000 to 3000 micrograms per 100 grams. In another embodiment, the dried fermented biomass show a vitamin K2 concentration ranges from 100 to 150 micrograms per 100 grams.

[0113] In any of the embodiments of the present invention, the term “vitamin K2” and “menaquinone” (abbreviated as MK-n, where "n" represents the chain length) is used interchangeably herein. It includes a range of isoforms, specifically MK-4, MK-5, MK-6, MK-7, MK-9, MK-10, MK-11 , MK-12, MK-13 and MK-14, with MK-7 being the most important and preferred isoform.

[0114] Any reference to 'an' item refers to one or more of those items. The term 'comprising' is used herein to mean including the method blocks or elements identified, but that such blocks or elements do not comprise an exclusive list and an apparatus may contain additional blocks or elements and a method may contain additional operations or elements. Furthermore, the blocks, elements and operations are themselves not impliedly closed.

[0115] The steps of the methods described herein may be carried out in any suitable order, or simultaneously where appropriate. The arrows between boxes in the figures show one example sequence of method steps but are not intended to exclude othersequences or the performance of multiple steps in parallel. Additionally, individual blocks may be deleted from any of the methods without departing from the spirit and scope of the subject matter described herein. Aspects of any of the examples described above may be combined with aspects of any of the other examples described to form further examples without losing the effect sought. Where elements of the figures are shown connected by arrows, it will be appreciated that these arrows show just one example flow of communications (including data and control messages) between elements. The flow between elements may be in either direction or in both directions.

[0116] Where the description has explicitly disclosed in isolation some individual features, any apparent combination of two or more such features is considered also to be disclosed, to the extent that such features or combinations are apparent and capable of being carried out based on the present specification as a whole in the light of the common general knowledge of a person skilled in the art, irrespective of whether such features or combinations of features solve any problems disclosed herein. In view of the foregoing description it will be evident to a person skilled in the art that various modifications may be made within the scope of the invention.EXAMPLES

[0117] In the following, non-limiting experimental examples in accordance with the invention are described. In accordance with the present invention, the method for producing fermented products with enhanced vitamin K2 content includes the following fermentation step:

[0118] STEP 1 : Preparation of Seaweed Meal. The preparation of seaweed meal utilizing semi-dried seaweed fresh raw material is detailed in the following example, specifying the precise composition and ratios of ingredients used in the process.

[0119] Example 1 : Semi-Dried Seaweed

[0120] Example 1A:Semi-dried seaweed: 100 Kg (moisture content of 35-44%);Sugar: 11 .5 Kg of Sucrose or table sugar;Yeast: 1.2 Kg of yeast (Saccharomyces cerevisiae and Candida utilis at a ratio of 1:0.2- 1);Enzyme: 0.46 Kg of Cellulase;Water: 58 Liters.

[0121] Example 1 B:Semi-dried seaweed: 100 Kg (moisture content of 45-55%);Sugar: 10 Kg of Sucrose or table sugar;Yeast: 1 Kg of Baker’s yeast (Saccharomyces cerevisiae);Enzyme: 0.4 Kg of Cellulase;Water: 50 Liters.

[0122] Example 1C:Semi-dried seaweed: 100 Kg (moisture content of 56-65%);Sugar: 8 Kg of Sucrose or table sugar;Yeast: 0.8 Kg of yeast (Candida utilis);Enzyme: 0.32 Kg of Cellulase;Water: 40 Liters.

[0123] Example 2: Fresh Seaweed

[0124] Example 2A:Fresh seaweed: 350 Kg, harvested within 1 to 5 days (moisture content 85%-92%);Sugar: 10 Kg Sucrose or table sugar;Yeast: 1 Kg of Baker’s yeast (Saccharomyces cerevisiae);Enzyme: 0.4 Kg of Cellulase;Water: 20 Liters.

[0125] Example 2B:Fresh seaweed: 350 Kg, harvested within 1 to 5 days (moisture content 93%-98%);Sugar: 5 Kg Sucrose or table sugar;Yeast: 0.5 Kg of Baker’s yeast (Candida utilis);Enzyme: 0.2 Kg of Cellulase;Water: 10 Liters.

[0126] Example 2C:Fresh seaweed: 350 Kg, harvested within 1 to 5 days (moisture content 93%-98%);Sugar: 5 Kg Sucrose or table sugar;Yeast: 0.5 Kg of yeast (Saccharomyces cerevisiae and Candida utilis at a ratio of 1 :0.2- 1);Enzyme: 0.2 Kg of Cellulase.Water: 10 Liters.

[0127] STEP 2: Preparation of Yeast Culture

[0128] Mixing Ingredients: Combine sugar, yeast, enzyme, and water in a sterile container.

[0129] Dissolution: Stir the mixture thoroughly until all components are fully dissolved.

[0130] Incubation: Allow the inoculant to develop by preparing it at least 6 hours in advance, but no more than 32 hours before combining with the seaweed raw material.

[0131] STEP 3: Washing of Seaweed

[0132] Submerge the semi-dried or fresh seaweed in fresh water for a duration of 2 to 5 hours. As a result of this step, the contaminants and debris are removed from the seaweed, ensuring a clean raw material for fermentation.

[0133] STEP 4: Blanching (Applicable for Semi-Dried Seaweed Only)

[0134] Semi-dried seaweed is blanched in hot water at a temperature of more than 85°C and for a duration of 15 to 35 minutes. For the fresh seaweed raw material, blanching is not necessary.

[0135] STEP 5: Size Reduction

[0136] The seaweed material is minced with a meat grinder or a similar cutting tool to archive a material size ranging between 3 mm and 8 mm to optimize fermentation efficiency.

[0137] STEP 6: Mixing with Fermentation Inoculant

[0138] The seaweed material is mixed with the fermentation inoculant from step 2. In this phase, filtered water is added to the mixture until it reaches a consistency that allows easy stirring with a paddle. This agitation of the mixture can be achieved either using a mechanical paddle or pulsating air.

[0139] STEP 7: Fermentation Process

[0140] The mixture is transferred into a fermentation tank, where it constitutes the biomass.

[0141] In said fermentation tank a mixing or agitation phase is performed at least once every 24 hours to maintain homogeneity of the biomass.

[0142] In this phase, the pH level is measured at 12-hour intervals, and the fermentation is considered complete when the pH reaches a range between 3.5 and 4.5.

[0143] STEP 8: Post-Fermentation Processing

[0144] In the post-fermentation phase, the liquid component is extracted from the biomass by straining or draining it from the fermentation tank, and the remaining solid biomass is transferred to a drying facility for further processing.

[0145] The extracted liquid is collected and subjected to dehydration, utilizing a spray dryer or a freeze dryer.

[0146] The residual solid biomass is transferred to a drying facility, where it is dried using one of the following methods: a heat pump oven, a freeze drier, or a solar dryer.

[0147] After the drying process, the biomass is processed further through grinding, to achieve a fine, uniform product, followed by packaging for distribution or storage.

[0148] The fermented products obtained through the present invention exhibit significantly improved vitamin K2 content. These final products are available in various forms to cater to diverse applications: Extracted Liquid: A concentrated liquid rich in vitamin K2, derived directly from the fermentation process. This vitamin-rich extract is ideal for incorporation into beverages, nutritional supplements, or as a fortifying ingredient in food products.

[0149] Fermented Solid Biomass: The solid biomass resulting from fermentation, enriched with elevated levels of vitamin K2. This form can be utilized directly as a functional food ingredient, animal feed additive, or further processed for specific applications requiring a solid medium.

[0150] Dried Fermented Biomass: The fermented biomass that has been thoroughly dried, ensuring extended shelf life and ease of handling. Retaining its enhanced vitamin K2 content, this form is suitable for use in powdered supplements, tablets, capsules, or as an additive in various nutraceutical formulations.

[0151] The products obtained through the present invention are suitable for humans and / or animals, with a preference for utilization in the food intended for human consumption. Advantageously, incorporating the disclosed fermentation products into food enhances their nutritional value by increasing the vitamin K2 content. This enrichment of vitamin K2 contributes to the dietary intake of individuals, supporting in particular the bone health.

[0152] Table 1 : Endemic bacterial population of Bacillus spp. isolated from the Kappaphycus alvarezii seaweed used in the fermentation process of the present invention.

[0153] Table 2: Vitamin K2 composition in the fermented product. Data from three separate consecutive batches.

[0154] Various publications, patents, and / or references have been cited herein, the contents of which, in their entirety, are incorporated herein by reference. Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0155] Although the present invention has been described in detail with regard to certain preferred methods, other embodiments, versions, and modifications within the scope ofthe present invention are possible. Accordingly, the spirit and scope of the following claims should not be limited to particular descriptions of the embodiments set forth above.

Claims

CLAIMS1. A method for obtaining one or more products with a content of vitamin K2, the method comprising:(a) inoculating an algae biomass with a yeast strain and a carbohydrate source; and(b) performing a fermentation process on the algae biomass inoculated with a yeast strain and a carbohydrate source, wherein the fermentation process is performed in the presence of one or more Bacillus strains to increase the production of vitamin K2; wherein the one or more Bacillus strains are naturally occurring on the algae or isolated cultures.

2. The method according to claim 1, wherein the method further comprises performing an enzymatic treatment of the algae biomass material at a temperature between 30°C and 40°C.

3. The method according to any one of claims 1 or 2, wherein the method further comprises inoculating the enzymatically treated algae biomass with at least one yeast strain in the presence of a carbohydrate source.

4. The method according to any one of claims 1-3, wherein the method further comprises performing a fermentation process on the algae biomass at a pH between 6.5 and 4.0.

5. The method according to any one of claims 1-4, wherein the method further comprises recovering one or more fermentation products from the fermentation process.

6. A method for increasing the quantity of vitamin K2 obtained by fermenting algae biomass, the method comprising:(a) performing an enzymatic treatment of the algae biomass material at a temperature between 30°C and 40°C;(b) inoculating the enzymatically treated algae biomass with at least one yeast strain in the presence of a carbohydrate source;(c) performing a fermentation process on the algae biomass at a pH between 6.5 and 4.0;(d) recovering one or more fermentation products from the fermentation process; wherein the fermentation process is performed in the presence of one or more Bacillus strains capable of producing and / or enhancing the production of vitamin K2, wherein said one or more Bacillus strains are naturally present on the algae or isolated cultures.

7. The method according to any one of claims 1-6, wherein the one or more Bacillus strains are selected from the group consisting of Bacillus pumilus, Bacillus altitudinis, and Bacillus subtilis.

8. The method according to any one of claims 1-7, wherein the products have a higher content of vitamin K2 than a product or products obtained from a method comprising a fermentation process performed on an algae biomass in the absence of a yeast strain and one or more Bacillus strains.

9. The method according to any one of claims 1-8, wherein each of the one or more products are independently in the form of liquid extract, fermented solid biomass, or dried fermented biomass.

10. The method according to any one of claim 1-9, wherein the one or more products are in the form of liquid extract.

11. The method according to any one of claim 1-9, wherein the one or more products are in the form of dried fermented biomass.

12. The method according to any one of claim 1-9, wherein the one or more products are in the form of fermented solid biomass.

13. The method according to any one of claims 1-12, wherein the algae material is a seaweed material.

14. The method according to any one of claims 1-13, wherein the algae biomass is selected from the group consisting of Kappaphycus spp, Ascophyllum nodosum, Fucus vesiculosus, Laminaria japonica, Ulva lactuca, Sargassum spp., Porphyra spp., Eucheuma spp., Macrocystis pyrifera (commonly known as giant kelp), Gracilaria spp.

15. The method according to claim 14, wherein the algae biomass is Kappaphycus alvarezii.

16. The method according to any one of claims 1-15, wherein the one or more Bacillus strains are isolated from algae material.

17. The method according to claims 1-16, wherein the method further comprises isolating the one or more Bacillus strains from algae material.

18. The method according to claim 17, wherein the isolating the one or more Bacillus strains from algae material comprises:(a) collecting algae material from different sites and placing them in sterile containers;(b) treating the algae material with ethanol, followed by rinsing with sterile seawater;(c) homogenizing and cut in small piece the algae material, in sterile seawater;(d) performing serial dilutions of the homogenate and plating aliquots onto marine agar to culture bacteria;(e) incubating the plates to allow bacterial colonies to grow;(f) selecting and purifying individual colonies to obtain pure Bacillus strains.

19. A method for producing vitamin K2, comprising: a) implementing the method according to any one of claims 1-18; and, b) recovering the vitamin K2.

20. The method according to any one of claims 1-19, wherein the fermentation process is performed in a fermentation tank.

21. The method according to any one of claims 1-10 or 13-20, wherein the liquid extract exhibits a vitamin K2 concentration ranging from 1,000 to 1 ,100 micrograms per 100 grams.

22. The method according to any one of claims 1-9 or 11 or 13-20, wherein the dried biomass exhibits a vitamin K2 concentration ranging from 100 to 150 micrograms per 100 grams.

23. The method according to any one of claims 1-9 or 12-20, wherein the fermented seaweed solid biomass in its wet form exhibits a vitamin K2 concentration ranging from 2,000 to 3,000 micrograms per 100 grams.

24. Use of one or more isolated Bacillus strains for obtaining vitamin K2 from algae biomass.

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