Process for producing plant-based fermented food or feed with umami-rich sensory profile, reduced off flavor and multifunction, products or ingredients obtained therefrom and uses thereof

Abstract

The present invention relates to a process for producing plant-based fermented food or feed containing plant protein with umami-rich sensory profile and improved nutritional profile, as well as products obtained therefrom and uses thereof. In particular the present invention relates to a process for producing fermented food or feed wherein the plant proteins are cross-fermented by the use of the combination of Propionibacterium spp., Lactobacillus spp., Aspergillus spp., and Saccharomyces spp. as well as products obtained therefrom and uses thereof. The process of the invention is based on the use of solid-state-fermentation (SSF) and submerged fermentation (SMF).

Description

[0001] Process for producing plant-based fermented food or feed with umamirich sensory profile, reduced off flavor and multifunction, products or ingredients obtained therefrom and uses thereof.

[0002] Technical field of the invention

[0003] The present invention relates to a process for producing plant-based fermented food or feed containing plant protein with umami-rich sensory profile, reduced off flavor and multifunction, as well as products obtained therefrom and uses thereof. In particular the present invention relates to a process for producing fermented food or feed wherein the plant proteins are cross-fermented by the use of the combination of Propionibacterium spp., Lactobacillus spp., Aspergillus spp., and Saccharomyces spp. as well as products obtained therefrom and uses thereof.

[0004] The process of the invention is based on the use of solid-state-fermentation (SSF) and submerged fermentation (SMF).

[0005] Background of the invention

[0006] The rising prominence of diets among adults and children is underscored by growing concerns over environmental sustainability and long-term health outcomes, including reduced risks of cancer and cardiovascular diseases. While such diets offer numerous health and nutritional benefits, a significant portion of these advantages stems from the absence of animal products, which often contains large amounts of e.g. unhealthy saturated fats.

[0007] However, plant-based diets also present certain nutritional challenges when compared to their animal-based counterparts. Critical nutrients like vitamin B12 are absent in unfortified plant-based foods, and phytic acids, commonly found in whole grains and legumes, hinder proper mineral absorption.

[0008] In addition to nutritional disparities, a significant obstacle hindering broader acceptance of plant-based diets is the perceived taste of plant-based products, which remains the primary deterrent for consumers in adopting such dietary choices (Smart Protein Project EU, 2023). Despite extensive efforts by the industry to enhance organoleptic properties, consumer satisfaction and adoption rates continue to lag behind expectations (Wilson C., Food Business News, 2023).

[0009] A key contributing factor to this discrepancy may lie in the historical separation among functional nutritional ingredients, texturants and flavor additives. Traditionally, flavor components have been employed primarily to mask off-notes and enhance overall palatability, albeit with limited success. However, to achieve truly appealing sensory qualities, the flavors inherent to the nutritional ingredients themselves must play a more integral role, given their predominant presence in the overall food composition consumed.

[0010] Moreover, limitations exist with animal-free analogues, presenting challenges for a substantial portion of consumers (Rees T., Euromonitor International, 2023). The differences in molecular and physicochemical properties between plant-derived and animal-derived ingredients make it difficult to replicate the texture of animal products using plant-based ingredients (Xia, YJ, et al. 2023). The attempt to replicate both the flavor and texture of animal-derived products can often backfire, either by coming across as inauthentic or by failing to meet consumer expectations for a genuine plant-based culinary experience. Consequently, the flavor and umami profile needed to elevate plant-based foods should stand apart from meat-based analogues. Instead, the focus should be on accentuating the inherent flavors of plant ingredients, thereby promoting the adoption of a plantrich diet for future sustainability and health.

[0011] To date, existing solutions have predominantly addressed specific nutritional deficiencies or sensory challenges in isolation, targeting individual aspects such as protein, iron, vitamin B12, or flavor enhancement. However, none have delivered a comprehensive solution encompassing nutritional, sensory and multifunction considerations. Furthermore, no method has been devised to harness the potential synergy of cross-fermentation, a sequential order of fermentation processes, and the dual application of solid-state fermentation and submerged fermentation to achieve a holistic enhancement in both nutrition, sensory and function attributes. While noteworthy examples exist, demonstrating progress in this field, a definitive solution that addresses the multifaceted aspects of nutrition and sensory appeal remains elusive. Cross-fermentation, which also can be referred to as sequential fermentation, has historical precedence in traditional fermentation practices, characterized by the spontaneous involvement of fungi, bacteria, and yeast. This phenomenon is observed in various fermented foods and beverages, including wine production, where the utilization of indigenous fungal and yeast strains contributes significantly to the development of distinctive flavor profiles (Liu D. et al., 2020; Cubillos F. et al., 2019).

[0012] In the realm of fermentation studies, some experiments have explored crossover fermentation, involving the combination of strains and substrates from established product processes. One notable example is the Dairy Miso Case study (Dank A. et al., 2021).

[0013] Protein availability and sensory attributes

[0014] Xiang H, et al., 2019 highlights how fermentation enhances the nutritional profile of foods by increasing the bioavailability of nutrients and synthesizing beneficial metabolites such as vitamins and amino acids.

[0015] Clark A. J. et al., 2022, discloses that shiitake mycelium fermentation improves digestibility, nutritional value, flavour and functionality of plant proteins while US20200245640A1 discloses the use of enzymes to deflavor pea protein.

[0016] Similarly, US8153174B2 discloses the outcome as a 'deflavoring' process rather than the creation of specific desirable flavors. Moreover, while enhancing the Protein digestibility-corrected amino acid score (PDCAAS)through methods like hydrolysis is established, conventional approaches have relied on synthetic enzymes, often resulting in heightened bitterness perception. Hydrolyzed Plant Protein (HVP) has been engineered to mimic meat-like sensory profiles; however, the associated process has been linked to potential carcinogenicity concerns. Critically, HVP yields a product with sodium levels exceeding 30% dry weight, rendering it unsuitable as a nutritional enhancement ingredient.

[0017] Texture function Texture of plant based food is one of major challenge which lower consumer acceptance and satisfaction. Fermentation can modify the texture properties of plant proteins. WO2023126389 discloses one effective method which involves solid fermentation using mycelia of filamentous fungi and transforms plant substrates into compositions. This process is particularly useful for creating meat analogues, as the fungal growth imparts structural changes that result in a fibrous texture similar to that of meat.

[0018] Microbial safety with low sodium

[0019] Conventional fermented foods often necessitate high salt concentrations (> 10%) to ensure microbiological safety throughout the production process. However, this elevated salt content, which can reach >20-40% in powdered form postprocessing, renders the product unsuitable for widespread use as a macronutritional enhancer, as consumption quantities would likely exceed daily sodium intake limits. While salt-free alternatives exist, they require complex processing setups and the addition of known preservatives like nisin, being a polycyclic antibacterial peptide produced by the bacterium Lactococcus lactis.

[0020] CN1935009B e.g. discloses a method for producing rapid fermented type salt-free miso-like food material with favourable taste and flavor.

[0021] Vitamin Bl 2

[0022] Propionibacterium freudenreichii is a well-established bacterium known for its capacity to synthesize vitamin B12 through fermentation (Deptula P. et al., 2017). However, its origin from dairy sources renders it unsuitable for incorporation into plant-based foods.

[0023] Hence, in view of the prior art disclosures, an improved fermentation process for producing plant-based food products with both high nutritional qualities and sensory value would be advantageous.

[0024] Summary of the invention

[0025] The present invention relates to an improved process for producing fermented, plant-based food products and feed ingredients with both high nutritional qualities, improved texture functions and sensory value. Moreover, the process of the present invention addresses the limitations in the prior art by creating a microbial-safe plant-based product with minimal to no salinity levels. Even further, the process of the present invention is based on non-dairy culture enabling the natural production of vitamin B12, integrating said culture into high- quality plant proteins, thereby imbuing them with enhanced nutritional value while ensuring sensory appeal.

[0026] Hence, one objective of this invention is to produce a plant-based food product or feed ingredient with, amongst other, vitamin B12-enrichment, low salinity elevated nutritional qualities, and texture functions while being a product exhibiting robust sensory characteristics.

[0027] Specifically, the synergistic effects of the process of the present invention manifest across four key aspects:

[0028] Sensory enhancement

[0029] The fermentation process of the present invention effectively eliminates undesirable sensory qualities commonly associated with plant proteins, such as beany notes, green undertones, and bitterness, while concurrently enhancing desirable sensory attributes like umami richness, flavor depth, and complexity. Additionally, improvements in mouthfeel are notable, including the elimination of grittiness and reduced mouth-drying effects.

[0030] Nutritional advancements

[0031] The food product or feed ingredient of the present invention boasts an enhanced nutritional profile unattainable through conventional means. Notable improvements include elevated levels of vitamin B12 compared to plant-derived sources, alongside increased concentrations of essential minerals such as magnesium. Furthermore, the inclusion of post-biotics, coupled with fibre / pre- biotics, contributes to gut health support.

[0032] Quality and stability enhancement

[0033] The fermentation process of the present invention not only improves the quality of the resultant product but also enhances its stability without the need for additional food preservatives or pH adjustments during fermentation. This ensures a product of superior quality and a more natural process. Functional property optimization

[0034] The product exhibits improved functional protein properties that traditionally have been challenging to achieve with plant proteins. These include enhanced solubility in liquid mediums and minimal thickening and clumping tendencies when subjected to heat treatment, exhibiting improved emulsion and creaminess ability thereby enhancing its versatility in various food applications.

[0035] The objectives of the present invention are achieved through the synergistic interplay of various microorganisms, including bacteria, yeasts, and fungi, alongside a diverse array of legumes or grains utilized as substrates in a dualstage fermentation process utilizing a combination of solid-state-fermentation (SFF) and submerged fermentation (SMF) with a sequential order.

[0036] Thus, one aspect of the invention relates to a process for producing a fermented plant-based food product or feed ingredient, the process comprising the steps of: a) mixing legume, whole grain bran and / or seaweed and water, b) heating and subsequently cooling of the mixture resulting from step a), c) inoculating the material resulting from step b) with cl) whole grain solid-state fermented (SSF) in the presence of one or more Aspergillus spp. strains, c2) and one or more Propionibacterium spp. strains, d) first submerged fermenting (SMF) the mixture from step c) in a closed system without stirring, e) inoculating the material resulting from step d) with el) one or more Lactobacillus spp. strains and e2) one or more Saccharomyces spp. strains, f) second submerged fermenting (SMF) the mixture from step e) in a closed system with stirring, g) heating and subsequently cooling of the mixture resulting from step g), h) drying the mixture resulting from step f), thereby providing a plant-based fermented food product or feed ingredient with umami-rich sensory profile and multifunction. Brief description of the figures

[0037] Figure 1 shows an overview of the steps to be performed in relation to the process of the present invention, i.e. solid-state-fermentation (SSF), first and second stages of submerged fermentation (SMF), the steps of mixing, heat treatment, cooling, inoculation, first stage SMF-fermentation, second stage SMF- fermentation, heat treatment, cooling and downstream processing.

[0038] Figure 2 shows an overview of dynamics cross feeding and metabolites exchange in relation to the cross fermentation process of the present invention. Each cross fermentation step contributes to flavour enhancement, nutritional and functional enrichment.

[0039] Figure 3 Principal Component Analysis (PCA) of semipolar metabolites across different fermentation processes, including cross fermentation, only first submerged fermentation stage, only second submerged fermentation stage, all microorganisms involved in both first and second submerged fermentation stages.

[0040] Figure 4 Principal Component Analysis (PCA) biplot of SCFAs, amino acids, and TCA cycle metabolites across different fermentation processes, including cross fermentation, only first submerged fermentation stage, only second submerged fermentation stage, all microorganisms involved in both first and second submerged fermentation stages.

[0041] Figure 5. Principal Component Analysis (PCA) of sensory attributes for four ingredient samples applied in a risotto with mushroom broth: 215 (Cross fermentation), 589 (Raw material), 401 (Inactive step between two submerged fermentation stages), and 745 (Only second submerged fermentation stage). The PCA plot shows the distribution of samples based on consumer-rated sensory descriptors, including umami, saltiness, creaminess, bitterness, aroma liking, taste liking, off-aroma, thickness, and continuity.

[0042] Figure 6. Principal Component Analysis (PCA) of sensory attributes for the four ingredient samples (147 - Cross fermentation sample, 354 - Raw material sample, 910 - Inactivation between two submerged stages sample, 842 - Second submerged fermentation stage sample). The plot displays the distribution of samples based on consumer-rated sensory attributes, including umami, creaminess, viscosity, bitterness, slipperiness, graininess, and off-flavor.

[0043] The present invention will now be described in more detail in the following.

[0044] Detailed description of the invention

[0045] The present invention relates to a process for producing a fermented plant-based food product or feed ingredient.

[0046] Further, the present invention relates to fermented plant-based food products or feed ingredients with increased umami, improved nutrition and sensory qualities developed from bacteria, yeasts, and fungi, wherein the protein content is 40- 80% dry matter, the fibre content is 10-30g / 100g dw, the iron content is 4- 15mg / 100g dw, and the vitamin B12 content is 5-25pg / 100g dw

[0047] Specifically, the process of the present invention provides fermented plant-based food products or feed ingredients:

[0048] • with an improved protein bioavailability that Protein digestibility-corrected amino acid score (PDCAAS) of above 0.75,,

[0049] • where the score of bitterness (phenols, flavonoids, isoflavones, terpenes, and glucosinolates) is <3 in a sensory test on a point scale of 1-5, i.e. from low to undetectable in the food product or feed ingredient,

[0050] • where the score of the savory umami profile is >4 in a sensory test on a point scale of 1-5 and glutamate is >350mg per 100g dw,

[0051] • where the mouthfeel (creaminess, slipperiness, smoothness) is >3.5 in a sensory test on a point scale from 1-5, and

[0052] • where the level of graininess, beany flavour (unsaturated fatty acids) decreases compared to a corresponding unfermented product / ingredient, score<1.8 in a sensory test on a point scale from 1-5,

[0053] The process of the present invention further provides a microbial-safe food product or feed ingredient with no detectable aflatoxins, Bl, B2, Gl, G2, deoxynivalenol (vomitoxin), zearalenone (ZON), T-2 toxin, HT-2 toxin, ochratoxin A., Enterobacteriaceae not calculable, i.e. < 10 CFU / g, moulds and yeast not calculable, i.e. < 100 cfu / g, aerobic plate count (APC) <10,000 cfu / g.

[0054] The present invention also provides increased umami of a fermented plant-based food product or feed ingredient according to the invention.

[0055] The present invention even further provides fermented plant-based food products or feed ingredients with a content of umami (glutamic acid) of 6-9 g / lOOg and free glutamic acid of calculated as a monosodium glutamate (MSG) content of 400-570 mg / 100g dw, compared to a fermented but not cross fermented variant of >270 mg / lOOg dw.

[0056] The present invention relates to improved nutrition of a fermented plant-based food product or feed ingredient with improved nutrition.

[0057] The present invention furthermore provides fermented plant-based food product or feed ingredient with improved sensory and / or organoleptically sensations.

[0058] Definitions

[0059] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0060] Dry weight

[0061] When used herein, dry weight (dw) refers to that water / moisture has been removed to the degree possible (3-6% moisture in finish material) and the substance of the material is dry and solid, thereby enabling a more accessible point of reference when comparing compounds or elements.

[0062] Seaweed

[0063] When used herein, the term seaweed refers to edible seaweeds, such as kelp, nori, duckwheat, wakame, hijiki, kombu, chlorella, spirulina, sea moss and similar.

[0064] First and second stage SMF-fermentation When used herein and with specific reference to figure 1, first stage submerged fermenting (SMF) refers to the SMF performed in step c) of the claimed process, i.e. in a closed system without stirring, whereas the second stage submerged fermenting (SMF) refers to the SMF performed in step f) f) of the claimed process, i.e. in a closed system with stirring.

[0065] Solid-state- fermentation (SFF)

[0066] When used herein, the term solid state fermentation (SSF) refers to a biomolecule manufacturing process used e.g. in the food industry. These biomolecules are mostly metabolites generated by microorganisms grown on a solid support selected for this purpose. This technology for the culture of microorganisms is an alternative to liquid or submerged fermentation, used predominantly for industrial purposes. Solid state fermentation uses cultures and substrates with low water levels (reduced water activity), which is particularly appropriate for mould.

[0067] Submerged fermentation (SMF)

[0068] When used herein, the term submerged fermentation (SMF) refers to the substrate being submerged in liquid has conditions that limits oxygen exposure, promoting anaerobic growth of desired microorganisms. Water or brine is a dominant component of the substrate.

[0069] Cross- fermentation

[0070] When used herein, the term "cross-fermentation" refers to processes in which a microorganism from one traditional fermentation process is introduced onto a new substrate and / or to a new partner. In addition, in the context, it also means the sequential use of SSF and SMF processes.

[0071] Koji

[0072] When used herein, Koji refers to whole grains or beans that has been inoculated grain solid-state fermented (SSF) in the presence of one or more Aspergillus species.

[0073] Miso When used herein, miso refers to a traditional Japanese seasoning. It is a thick paste produced by the fermentation of soybeans with the fungus Aspergillus oryzae and sometimes rice, barley, or other grains.

[0074] Pulse flour legumes

[0075] When used herein, pulse flour legumes refer to legumes ground to produce whole or dehulled flours. To produce a whole pulse flour, the entire seed can be milled as part of a continuous process or the hull can be collected as a co-product of dehulling, separately ground, then recombined with the flour at naturally occurring levels after milling.

[0076] Umami

[0077] When used herein, the term umami refers to one of the five basic tastes, along with sour, sweet, bitter, and salty, comes from three compounds that are naturally found in plants and meat: glutamate, inosinate, and guanylate. Glutamate is an amino acid found in vegetables and meat. Inosinate is primarily found in meat, and guanylate levels are the highest in plants.

[0078] VVM

[0079] When used herein, VVM refers to volume of air sparged (in aerobic cultures) per unit volume of growth medium per minute, it is calculated by dividing the measured airflow rate (units: L / m, using a rotameter) by the volume (L) of growth medium (including cultured cells). Hence, the first 'V stands for volume of air; the second 'V stands for per unit of medium; 'M' stands for per unit of time, for example 1 VVM (l / l / m) means in 1 minute time there is 1 litre of air passing through 1 litre of medium.

[0080] PCA

[0081] When used herein, PCA refers to principal component analysis, the analysis is often used to reduce the number of dimensions in large datasets to principal components that retain most of the original information.

[0082] SCFAs

[0083] When use herein, SCFAs refers to short chain fatty acids, which represents a group of fatty acids with 2 to5 carbon atoms. During certain fermentation condition, certain types and amounts of SCFAs could be produced by microorganisms by utilizing substrates. SCFAs have been claimed to have positive effects on the balance of gut microbiota to stimulate the gut health.

[0084] TCA

[0085] When used herein, TCA refers to tricarboxylic acid cycle, which is a series of biochemical reactions that happen at different stages of fermentation (either by anaerobic respiration or aerobic respiration) to release the energy stored in nutrients via the oxidation of acetyl-CoA derived from carbohydrates, fats, proteins, and alcohol.

[0086] Embodiments of the invention

[0087] One aspect of the present invention relates to a process for producing a fermented plant-based food product or feed ingredient with umami-rich sensory profile and multifunction, the process comprising the steps of: a) mixing legume, whole grain bran and / or seaweed and water, b) heating and subsequently cooling of the mixture resulting from step a), c) inoculating the material resulting from step b) with cl) whole grain solid-state fermented (SSF) in the presence of one or more Aspergillus spp. strains, c2) and one or more Propionibacterium spp. strains, d) first submerged fermenting (SMF) the mixture from step c) in a closed system without stirring, e) inoculating the material resulting from step d) with el) one or more Lactobacillus spp. strains and e2) one or more Saccharomyces spp. strains, f) second submerged fermenting (SMF) the mixture from step e) in a closed system with stirring, g) heating and subsequently cooling of the mixture resulting from step g), h) drying the mixture resulting from step f), thereby providing a fermented food product or feed ingredient.

[0088] In another aspects of the present invention relates to a process in which the following SSF pre-treatment steps i) and ii) are carried out before the mixing step a): i) inoculating koji mold spores collected from one or more Aspergillus species onto steamed grains like whole grain (e.g. barley, rice, brown rice, quinoa, oats, farro, spelt, buckwheat), ii) drying of the mixture resulting from step i).

[0089] Embodiments relating specifically to process step a)

[0090] Another aspect of the invention relates to a process for producing a fermented plant-based food product or feed ingredient with umami-rich sensory profile and multifunctional, the process comprising the steps of: a) mixing 10-40% (w / w) legume, 1-5% (w / w) whole grain bran and / or seaweed and 40-70% water, b) heating and subsequently cooling of the mixture resulting from step a), c) inoculating the material resulting from step b) with cl) whole grain solid-state fermented (SSF) in the presence of one or more Aspergillus spp. strains, c2) and one or more Propionibacterium spp. strains, d) first submerged fermenting (SMF) the mixture from step c) in a closed system without stirring, e) inoculating the material resulting from step d) with el) one or more Lactobacillus spp. strains and e2) one or more Saccharomyces spp. strains, f) second submerged fermenting (SMF) the mixture from step e) in a closed system with stirring, g) heating and subsequently cooling of the mixture resulting from step g), h) drying the mixture resulting from step f), thereby providing a fermented food product or feed ingredient.

[0091] In another embodiment, the legumes used in step a) of the present invention is selected from preferably a group consisting of faba bean, pea, soy bean, lupine, black beans, navy beans, chickpeas, lentils or combinations thereof.

[0092] In still another embodiment, the legumes used in step a) of the present invention is preferably in powdered form, such as legume flour, or pulse flour legume, i.e. made by grinding or milling whole pulses into flour, and nothing is removed during the milling process. In still another embodiment, the whole grain bran used in step a) of the present invention is preferably selected from a group consisting of rice, oat, wheat, rye or combinations thereof.

[0093] In still another embodiment, the seaweed in step a) is selected from kelp, nori, duckwheat, wakame, hijiki, kombu, chlorella, spirulina, sea moss or combinations thereof.

[0094] In still another embodiment, salt, preferably sea salt, may be added to the mixture in step a) in an amount of 0-7% (w / w).

[0095] Embodiments relating specifically to process step b)

[0096] Another aspect of the invention relates to a process for producing a fermented plant-based food product or feed ingredient, the process comprising the steps of: a) mixing 10-40% (w / w) legume, 1-5% (w / w) whole grain bran and / or seaweed and 40-70% water, b) heating at around 90-100°C for 5-30 min and subsequently cooling at a temperature of 35-40°C. of the mixture resulting from step a), c) inoculating the material resulting from step b) with cl) whole grain solid-state fermented (SSF) in the presence of one or more Aspergillus spp. strains, c2) and one or more Propionibacterium spp. strains, d) first submerged fermenting (SMF) the mixture from step c) in a closed system without stirring, e) inoculating the material resulting from step d) with el) one or more Lactobacillus spp. strains and e2) one or more Saccharomyces spp. strains, f) second submerged fermenting (SMF) the mixture from step e) in a closed system with stirring, g) heating and subsequently cooling of the mixture resulting from step g), h) drying the mixture resulting from step f), thereby providing a fermented food product or feed ingredient.

[0097] Embodiments relating specifically to process step c) In another embodiment, the whole grain in step cl) is selected from barley, rice, brown rice, quinoa, oat, farro, spelt, buckwheat or combinations thereof.

[0098] In still another embodiment, the one or more Aspergillus spp. strains used in step cl) is / are selected from a group consisting of Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii, Aspergillus luchuensis, Aspergillus sojae, Aspergillus luchuensis awamori, Aspergillus tamariior or combinations thereof.

[0099] In still another embodiment, the solid-state-fermented whole grain in step cl) is mixed with the mixture resulting from step b) in a weight ratio percentage of 5- 25% (w / w).

[0100] In still another embodiment, the one or more Propionibacterium spp. strains in step c2) is / are mixed with the mixture resulting from step cl) to a level of 1-10 log cfu / g.

[0101] In still another embodiment, the one or more Propionibacterium spp. strains in step c2) is / are selected from the group consisting of P. freudenreichii, P. thoenii, P. jensenii, and P. acidipropionici or combinations thereof.

[0102] Embodiments relating specifically to first SMF process step d)

[0103] Another aspect of the invention relates to a process for producing a fermented plant-based food product or feed ingredient, the process comprising the steps of: a) mixing 10-40% (w / w) legume, 1-5% (w / w) whole grain bran and / or seaweed and 40-70% water, b) heating at around 90-100°C for 5-30 min and subsequently cooling at a temperature of 35-40°C. of the mixture resulting from step a), c) inoculating the material resulting from step b) with cl) whole grain solid-state fermented (SSF) in the presence of one or more Aspergillus spp. strains, c2) and one or more Propionibacterium spp. strains, d) first submerged fermenting (SMF) the mixture from step c) carried out for 8-30 hours at 25-34°C, VVM of 0-0.5 and at a pH of 4.5-6 in a closed system without stirring, e) inoculating the material resulting from step d) with el) one or more Lactobacillus spp. strains and e2) one or more Saccharomyces spp. strains, f) second submerged fermenting (SMF) the mixture from step e) in a closed system with stirring, g) heating and subsequently cooling of the mixture resulting from step g), h) drying the mixture resulting from step f), thereby providing a fermented plant-based food product or feed ingredient with an umami-rich sensory profile and multifunctionality.

[0104] Embodiments relating specifically to process step e)

[0105] In another embodiment, the one or more Lactobacillus spp. strains in step el) is / are selected from the group consisting of Lactobacillus easel, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus easel, Lactobacillus brevis, Lactobacillus plantarum or combinations thereof.

[0106] In still another embodiment, the one or more Saccharomyces spp. strains in step e2) is / are selected from the group consisting of Saccharomyces pastorianus, Saccharomyces cerevisiae, Saccharomyces bayanus or combinations thereof.

[0107] In another embodiment, the 0.5-5 log efu / g of the one or more Lactobacillus spp. strains in step el) and 0.3-4% (w / w) of the one or more Saccharomyces spp. strains in step e2) are mixed with the mixture resulting from step d).

[0108] Embodiments relating specifically to process step f)

[0109] In another embodiment, the fermentation in step f) is carried out for 8-24 hours at 25-34°C, VVM of 0-0.5 and at a pH of 4.5-6.

[0110] Embodiments relating specifically to process step g)

[0111] In another embodiment, the heating in step g) is carried out at 90-100°C for 10- 40 min, and the subsequently cooling (till below 8°C) or alternatively downstream processing by e.g. spray-drying until a mixture with a water content of <8% is obtained. Embodiments relating to the dynamic feeding and the exchange of metabolites for a targeted profile of metabolites obtained by cross fermentation of the invention

[0112] In another embodiment, the present invention relates to the multi-stage fermentation process involves dynamic cross-feeding and metabolite exchange at sequential fermentations among Aspergillus spp., Propionibacterium spp., Saccharomyces spp., and Lactobacillus spp., each contributing to flavor enhancement, nutritional enrichment, and multifunctional properties. In the solid- state fermentation (SSF) stage, Aspergillus spp. secrete amylases and proteases, breaking down oat proteins and starches into fermentable sugars, free amino acids (such as glutamate), peptides, and nucleotides (IMP, GMP), which enhance umami. Additionally, organic acids produced during this stage help prime the environment for subsequent microbial growth. During the first submerged fermentation (SMF) stage, residual enzymes remain active, continuing to hydrolyze macromolecules. The sugars, amino acids, and organic acids generated in the SSF stage serve as nutrients for Propionibacterium spp., which metabolizes sugar and lactic acid into propionic and acetic acids, while simultaneously synthesizing vitamin B12. These metabolic activities further modify flavor and contribute to natural preservation. In the final microaerobic stage, Saccharomyces spp., and L. plantarum are added and they join the interactive mechanisms with others. Saccharomyces spp., fermentation generates nucleotides, aromatic compounds, intensifying umami and enhancing antioxidant properties. L. plantarum plays a crucial role in stabilizing vitamin B12, while also promoting additional B12 synthesis by Propionibacterium spp. Furthermore, Lactobacillus spp. and Propionibacterium spp. produce exopolysaccharides (EPS), which improve texture and contribute to mouthfeel.

[0113] Embodiments relating to products obtained by the process of the invention In another embodiment, the present invention relates to a fermented plant-based food product, such as plant protein, or a feed ingredient obtained by or obtainable by the process of the invention.

[0114] In still another embodiment, the present invention relates to a fermented plantbased food product or feed ingredient produced by a process according to the invention, having one or more of the following characteristics: (a) NaCI concentration lower than 5% w / w,

[0115] (b) glutamate content between 100 and 3500mg / 100g dw,

[0116] (c) vitamin B12 content of more than 5pg / 100g dw,

[0117] (d) protein content between 20 and 60 g / lOOg dw,

[0118] (e) Protein Digestibility Corrected Amino Acid Score) >75% increased EAA score 10-30%,

[0119] (f) phytic acid reduction of > 10-30%,

[0120] (g)Improved sensory profile and overall liking via enhanced flavor (umami, saltness) and texture (creaminess, slipperiness and smoothness) while reducing undesirable traits (off-flavor, bitterness and graininess).

[0121] In still another embodiment, the present invention relates to a fermented plantbased food product or feed ingredient produced by a process according to the invention, having one or more of the following characteristics: wherein said food product or feed ingredient comprises one or more of legumes, whole grain bran and / or seaweed and whole grain; wherein said food product or feed ingredient is having one or more of the following characteristics:

[0122] (a) protein content of 40-80% dry matter,

[0123] (b) fibre content of 10-30 g / lOOg dw,

[0124] (c) iron content of 4-15mg / 100g dw, and

[0125] (d) vitamin B12 content of 5-25pg / 100g dw.

[0126] In still another embodiment, the present invention relates to a fermented plantbased food product or feed ingredient produced by a process according to the invention, having one or more of the following characteristics:

[0127] (a) NaCI concentration lower than 2% w / w,

[0128] (b) glutamate content between 100 and 3500mg / 100g dw,

[0129] (d) vitamin B12 content of more than 5pg / 100g dw,

[0130] (e) iron content between 4 and 15mg / 100g dw,

[0131] (f) protein content between 40 and 80 g / lOOg dw

[0132] (g)PDCAAS (Protein digestibility-corrected amino acid score ) >75%, increased EAA score 10-30%,

[0133] (h) phytic acid reduction of >10-30%, and (i) Improved sensory profile and overall liking via enhanced flavor (umami, saltness) and texture (creaminess, slipperiness and smoothness) while reducing undesirable traits (off-flavor, bitterness and graininess).

[0134] In still another embodiment, the product may e.g. be a fermented plant-based food product or feed ingredient, produced according to the process of the present invention, having a glutamate content of 400-570 mg / lOOg dw.

[0135] In still another embodiment, the product may e.g. be a fermented plant-based food product or feed ingredient, produced according to the process of the present invention, having a DHA / EPA content between 200 and lOOOmg / lOOg dw, and / or a vitamin K content between 10 and 80pg / 100g dw.

[0136] Embodiments relating to use of the products according to the invention

[0137] In another embodiment, the present invention relates to the use of processes of fermenting plant-based materials comprising the process steps of the process of the invention for:

[0138] - the synthesis of vitamin B complex, preferably vitamin B12; and / or

[0139] - reducing the bitterness of plant protein hydrolysis; and / or

[0140] - improved sensory profile and overall liking via enhanced flavor (umami, saltness); and / or

[0141] - improved texture (creaminess, slipperiness and smoothness); and / or

[0142] - reducing undesirable traits (off-flavor and graininess) of raw plant ; and / or

[0143] - increasing content of DHA and / or EPA; and / or

[0144] - increasing the content of glutamic acid (umami); and / or

[0145] - increasing the mineral levels and bio-accessibility; and / or

[0146] - increasing the microbial safety; and / or

[0147] - increasing the PDCAAS (Protein Digestibility Corrected Amino Acid Score)); and / or

[0148] - reducing the phytic acid content; and / or

[0149] The present invention additionally relates to the following aspects:

[0150] • a method to naturally synthesize vitamin B12, through novel crossfermentation and sequential use of SSF and SMF in a plant-protein medium, and / or • a method to reduce the bitterness and deterrent flavor / aroma associated with plant proteins such as beanie, green and barn-like through plant protein hydrolysis using cross-fermentation of fungi, bacteria and yeast, and / or

[0151] • a method to increase glutamic acid (umami) in a plant-protein, vitamin B12 enhanced medium by cross-fermentation of fungi, bacteria and yeast, and / or

[0152] • a method to increase mineral levels and bio-accessibility through the synergistic effect of cross-fermentation of fungi and probiotics, and / or

[0153] • a method to achieve microbial safety by the synergistic effect of the media and inoculums with minimal sodium usage, and / or

[0154] • a method to enhance plant protein functionalities in application such as solubility and thermal stability, emulsification, fat binding and water binding effect along with improved mouthfeel such as reduced grittiness and mouth-drying effect and increased creaminess, slipperiness, smoothness.

[0155] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0156] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0157] The invention will now be described in further details in the following non-limiting examples.

[0158] Examples

[0159] Example 1A - the process of producing the "NUTRUMAMI product" (V49) Koji / solid-state-fermented whole grain of barley is prepared by inoculating koji mold spores collected from yellow Aspergillus oryzae onto steamed barley. It is then incubated under controlled conditions for 52 hours at temperatures 35°C and moisture levels of 42% Followed by a drying step at 42°C to moisture levels of <8%. 19% (w / w) of faba bean, 1.5% (w / w) whole grain bran (rice or oats) and 1.5% (w / w) salt, e.g. sea salt, is moistened with 70% water were homogenized / high shear blended at ambient temperature.

[0160] The slurry was heated to 100°C and held for 15 minutes. After cooling to 35°C, 9% (w / w) aforementioned koji / solid-state-fermented whole grain, 7 log cfu / g of Propionibacterium spp. were added.

[0161] The slurry was mixed and incubated in a closed system without stirring agitation for 18 hours at 29°C as first stage SMF-fermentation. Subsequently, 1 log cfu / g of Lactobacillus plantarum, and 1.2 % (w / w) of Saccharomyces pastorianus were added to the slurry at the beginning of the second stage SMF-fermentation under the microaerobic condition with stirring agitation for 10 hours at 29°C.

[0162] The product was then heat treated to a temperature at 92°C for 10 minutes followed by a cool down to < 8°C and directly transferred to the downstream processing, spray drying to produce a powder form product.

[0163] Example 1 - further aspects of the process of the invention

[0164] The fermented plant-based food product or feed ingredient according to the present invention may be produced by the following process.

[0165] Koji (solid-state-fermented whole grain, e.g. barley, rice, brown rice, quinoa, oats, farro, spelt, buckwheat) is prepared by inoculating koji mold spores collected from one or more Aspergillus species onto steamed grains like oat, rice or barley. It is then incubated under controlled conditions for 2 to 3 days at temperatures between 30°C to 40°C and moisture levels of 40% to 50%. Followed by a drying step at 40-50°C to moisture levels of <6%.

[0166] 10-40% (w / w) of legume / pulse flour (e.g. faba bean, pea, soy bean, lupine, black beans, navy beans, chickpeas, lentils), 1-5% (w / w) whole grain bran (e.g. rice, oats, wheat, rye) or seaweed (e.g. kelp, nori, duckwheat, wakame, hijiki, kombu, chlorella, spirulina, sea moss) and 0-7% (w / w) salt, e.g. sea salt, is moistened with 40-70% water were homogenized / high shear blended at ambient temperature.

[0167] The slurry was heated to 100°C and held for 5-30 minutes. After cooling to 35- 40°C, 5-25% (w / w) aforementioned koji / solid-state-fermented whole grain, 1-10 log cfu / g of Propionibacterium spp. were added.

[0168] The slurry was mixed and incubated in a closed system without stirring agitation for 8-30 hours at 25-34°C as first stage SMF-fermentation. Subsequently, 0.5-5 log cfu / g of Lactobacillus spp., and 0.5-4% (w / w) of yeast (e.g. Saccharomyces pastorianus, Saccharomyces cerevisiae and / or Saccharomyces bayanus etc.) were added to the slurry at the beginning of the second stage SMF-fermentation under the microaerobic condition with stirring agitation for 8-24 hours at 25-34°C.

[0169] The product was then heat treated to a temperature at 90-100°C for 10-40 minutes followed by a cool down to < 8°C and directly transferred to the downstream processing, which e.g. could be centrifugation, spray drying, hotfill, fluidized-bed dryer in order to provide a final product material in either liquid, paste or powder form.

[0170] Table 1: Composition of product

[0171] Example 2 - Choice of strains

[0172] The diverse combinations of various Propionibacterium spp. and Lactobacillus spp. together with multiple types of whole grain koji strain (i.e. whole grain that has been inoculated with one or more Aspergillus species) result in varying levels of vitamin B12 and sensory scores (see Examples 4 and 7) of the final products.

[0173] Two groups of Propionibacterium spp, three groups of Lactobacillus spp. and three groups of koji strains (i.e. whole grain that has been inoculated with one or more Aspergillus species) were included and tested to explore varying synergistic effects from both strain variations and log level of strains used in inoculation on the vitamin B12 levels and sensory scores of the final product (see Examples 4 and 7). The combination of Propionibacterium spp. and Lactobacillus spp. resulted in various vitamin B12 levels and sensory score of the product under the same fermentation process and condition (see Examples 4 and 7). a) P. freudenreichii , P. thoenii, P. jensenii, and P. acidipropionici b) Lactobacillus easel, Lactobacillus brevis, Lactobacillus plantarum c) The fermentation process contains one or more Propionibacterium strains from a) and one or more Lactobacillus strains from b)

[0174] The varying log levels of Propionibacterium spp. and Lactobacillus spp. resulted in various vitamin B12 levels and sensory scores of the product under the same fermentation process and condition (see Examples 4 and 7).

[0175] Different characteristic of koji and sensory profile via various koji strains

[0176] • Koji 1 Aspergillus oryzae Yellow (as per traditional classification standard in for example Japan)

[0177] • Koji 2 Aspergillus oryzae White Yellow (as per traditional classification standard in for example Japan)

[0178] • Koji 3 Aspergillus oryzae Black Yellow (as per traditional classification standard in for example Japan)

[0179] • Koji 4 Aspergillus sojae

[0180] • Koji 5 Aspergillus luchuensis awamori

[0181] • Koji 6 Aspergillus tamarii

[0182] Table 2: Characteristics, color, enzymatic activity and sensory profile using different Koji strains

[0183] Different types of koji strains with the same combination and log level of Propionibacterium spp. and Lactobacillus sp. resulted in various vitamin B12 levels and sensory score of the product under the same fermentation process and condition (see Examples 4 and 7).

[0184] Example 3- Test of different substrates

[0185] Multiple comparison tests were conducted on various substrates to identify optimal characteristics and assess the effects of the applied process across different substrates. Each substrate exhibited distinct yet perceptible sensory qualities, with all substrates demonstrating a positive enhancement in sensory attributes through the application of this process.

[0186] Significant findings emerged from tests conducted on legume substrates, with notable highlights as follows:

[0187] • Lentils: notably exhibited a rich savory umami profile reminiscent of charcuterie, accompanied by a robust color profile varying depending on the type of lentils tested.

[0188] • White beans: Showcased a pleasant, well-rounded umami profile with milder characteristics and an absence of typical "beany" sensory notes.

[0189] • Soy beans: Demonstrated a savory umami profile surpassing that of white beans, albeit lacking depth and sweetness observed in other substrates.

[0190] • Faba beans: Stood out with a heightened and well-balanced umami profile characterized by notable depth and sweetness, while maintaining a neutral color profile. Grain substrates were also subjected to evaluation in terms of both functional attributes and sensory qualities. A notable highlight is outlined as follows:

[0191] • Brown rice: Exhibited pleasant sensory characteristics with no detectable graininess, and a slightly less sweet profile compared to white rice or barley.

[0192] • White rice: Demonstrated neutral grain notes and a satisfying level of sweetness.

[0193] • Barley: Notable for the absence of barnyard notes and provided a pleasant nutty-sweet flavor profile.

[0194] • Oat: A unique twist to traditional koji, offering a nutty, creamy, and slightly sweet profile with a mellow umami depth.

[0195] Furthermore, each substrate enables the formulation of slightly different nutritional profiles, allowing for tailored formulations to emphasize specific nutritional focuses such as protein, iron, etc. This versatility in formulation offers the potential to address diverse dietary needs and preferences effectively.

[0196] In addition to the primary substrates, tests were conducted on supporting substrates that exert both nutritional and fermentative influences on the fermentation process. Notably, seaweed and algae were subjected to evaluation within the aforementioned process, yielding noteworthy results:

[0197] • Nori: Intensified umami flavors and depth, accompanied by a perceived increase in salinity and notable color impact.

[0198] • Kelp: While exhibiting less pronounced sensory impact than nori, kelp contributed to enhanced complexity in flavor.

[0199] • Spirulina: Surprisingly mitigated deterrent flavors associated with algae, such as earthiness and bitterness, while introducing sensory complexity and color enhancement.

[0200] Furthermore, rice and oat bran were evaluated, with no discernible differences in sensory attributes observed. However, heightened fermentation activity, characterized by excessive CO2 production, was noted, indicating potential fermentative benefits associated with these substrates.

[0201] Example 4 - Effect of vitamin B12 based on process conditions Commercial production of vitamin B12 through fermentation typically involves the utilization of buffer agents and synthetic nutrient mediums to achieve significant levels of vitamin B12 (>2pg / 100g w / w) necessary for regulatory claims. However, no previously described process has enabled the attainment of claimable vitamin B12 levels without the addition of such perceived less natural additives and processing.

[0202] The synergistic interaction of fungi, enzymes, bacterial cultures, and yeast according to the present invention creates an environment conducive to the production of vitamin B12 at claimable levels while still yielding a microbiologically safe end product. This effect, combined with carefully defined process conditions, optimizes vitamin B12 levels to exceed targeted claim levels and achieves production in a substantially shorter total fermentation time compared to existing commercial processes.

[0203] Medium

[0204] Table 3: Process conditions and effect on sensory score and vitamin B12 levels using different media

[0205] (Scale for "Overall Sensory Score": 1 Poor - 7 Excellent)

[0206] Rice bran (named "Bran" in table 2) at 1.5% led to an increase in vitamin B12 levels in two separate tests of 34 and 58% respectively. • Additions of Saccharomyces spp. (named "Yeast" in table 2) led to no decrease in vitamin B12 levels.

[0207] • The addition of Lactobacillus spp. (named "LAB" in table 2) earlier led to improved sensory and slightly slower drop of pH and increased vitamin B12 levels

[0208] Temperature

[0209] Table 4: Process conditions and effect on sensory score and vitamin B12 levels using different temperatures

[0210] (Scale for "Overall Sensory Score": 1 Poor - 7 Excellent)

[0211] Temperature above 29°C (tested variables 30°C and 32°C) led to a faster pH drop, in particular, the 32°C variant. Higher temperatures both improved but also could lower the vitamin B12 levels. Yet, a constant was poorer sensory score contributed to an observed difference in aroma and flavor profile due to difference in volatile compounds, but also sensory impact due to the change in pH for the 32°C variant. V31-A and V31-C were produced by the process of the invention except for the higher temperature and differenced in the scales of batches (V49 was produced in bigger quantities).

[0212] Process time

[0213] Table 5: Process conditions and effect on sensory score and vitamin B12 levels using different process times

[0214] (Table 5 continued)

[0215] (Scale for "Overall Sensory Score": 1 Poor - 7 Excellent) Shorter process times (8-14 hours) in the first stage of SMF-fermentation and longer (24-34 hours) in the second stage of SMF-fermentation resulted in a faster pH drop, reduced vitamin B12 levels, and reduced sensory score. Furthermore, medium (18-21 hours) and long (24-34 hours) in the first stage SMF-fermentation and medium (18-20 hours) to long (24-32 hours) in the second stage SMF-fermentation led to different results, each with key subtracting elements of one or more between low sensory score, perceived microbial unsafe (visible mould formation) and / or low vitamin B12 levels.

[0216] Medium length (18-21 hours) of first stage SMF-fermentation time and medium to short length (8-14 hours) of second stage SMF-fermentation time resulted in the best conditions.

[0217] Aeration / further conditions

[0218] High aeration (0.4-0.7 VVM) led to a faster pH drop and low vitamin B12 Low aeration (0.05-0.2 VVM) led to a slow pH drop but lower vitamin B12 levels microaerobic (<0.05 VVM) led to slowed pH drop and higher value vitamin B12

[0219] So, in view of the above observations is was concluded that a refined balance between the conditions of process time, temperature and VVM is critical to gain the best results in terms of sensorial, microbial and nutritional properties.

[0220] Example 5 - Protein and available essential amino acids

[0221] Plant-based proteins inherently exhibit lower bioavailability as well as not completed essential amino acids compared to animal-derived counterparts, posing a challenge for widespread adoption in dietary applications. Soy protein, while recognized as one of the most bioavailable among plant sources, is associated with allergenicity and consumer preferences that often lead to avoidance.

[0222] Moreover, enzymatic hydrolysis of plant proteins, which can increase digestibility, yields bitter peptides, primarily composed of hydrophobic amino acids, further complicating sensory acceptance and nutritional quality, both crucial factors for promoting a plant-centric diet.

[0223] The process according to the present invention addresses these challenges by employing solid-state fermentation of fungi to generate a distinctive enzyme family (proteinase) from commercially available hydrolytic enzymes. Additionally, the incorporation of specific microbial cultures and yeast strains synergistically enhances enzymatic activity, resulting in heightened hydrolysis efficacy, as visually observed. Comparative analysis against non-co-fermented variants reveals a substantial increase in essential amino acid profiles that impacts the PDCAAS (Protein digestibility-corrected amino acid score)indicating superior protein quality and bioavailability. In the present invention, Digestible Indispensable Amino Acid Ratio is also referred to as DIAAR.

[0224] Table 5

[0225] Example 6 - The distinct metabolite profiles from that of raw ingredients, individual fermentations, cross fermentation or fermentations involving all microorganisms in isolation."

[0226] Fermentation using individual microorganisms, or a single process involving all relevant microorganisms simultaneously, fails to produce the same diversity and specificity in metabolite profiles that contribute to taste, nutrition, and functional properties. To compare the metabolite profiles resulting from cross fermentation with those from other fermentation methods, both targeted metabolomics (focusing on short-chain fatty acids (SCFAs), amino acids, and tricarboxylic acid (TCA])cycle intermediates) and untargeted metabolomics were conducted.

[0227] Semipolar Metabolite Analysis method:

[0228] Conducted using UHPLC-MS (Vanquish LC and Orbitrap Exploris 240). Both positive and negative ion modes were used with polarity switching. Data were processed using Compound Discoverer, Skyline, and MATLAB-based pipelines. Metabolites were identified across four confidence levels based on retention time, mass accuracy (<3 ppm), and MS / MS spectra, using various public and in-house databases.

[0229] Semipolar Metabolite Profiling results:

[0230] Principal Component Analysis (PCA) of semipolar metabolites revealed clear differentiation between cross fermentation sample and all other sample types, including raw materials and staged fermentation processes (Figure 3). The first two components accounted for 83.25% of the total variance (PCI: 52.91%, PC2: 30.34%).

[0231] Cross fermentation sample clustered distinctly in the lower-right quadrant of the PCA plot, demonstrating a unique semipolar metabolite profile compared to both early-stage (1° and 2°) fermentations and the full microbial fermentation treatment. While all fermentation samples deviated from the raw material along PCI— reflecting microbial metabolic activity— cross fermentation showed the greatest separation, indicating the highest degree of biochemical transformation.

[0232] In contrast to the gradual progression observed from raw material to first and second stage fermentations, cross fermentation sample did not align with this trajectory, suggesting a qualitatively distinct metabolic outcome. Furthermore, its separation from the "Fermentation all microbes" treatment along PC2 highlights differences in metabolite composition potentially linked to controlled microbial consortia or targeted fermentation dynamics employed in the cross fermentation process.

[0233] These results confirm that cross fermentation induces a specific and differentiated semipolar metabolite signature, likely driven by its cross-fermentation process, supporting its role as a distinctive and more complex fermentation strategy among the tested conditions.

[0234] Short-Chain Fatty Acid (SCFA) Analysis method

[0235] Performed by GC-MS (Agilent 7890B GC with 5977B MS detector) using acidified samples and deuterium-labelled standards. Quality control included evaluation of matrix effects and analytical overlap, with peak deconvolution applied using PARADISe software.

[0236] Amino Acids and TCA Metabolites Panel method:

[0237] Conducted by GC-MS following derivatization with methyl chloroformate and addition of stable isotope-labelled standards. Data were quantified against external calibration curves and processed using Skyline and MATLAB pipelines with full QC checks.

[0238] SCFA, Amino Acids, and TCA Metabolite Profiling results:

[0239] Targeted profiling of short-chain fatty acids (SCFAs), amino acids, and TCA cycle intermediates was performed using GC-MS, and Principal Component Analysis (PCA) was applied to assess compositional differences across fermentation treatments. The first two principal components explained 86.76% of the total variance (PCI: 60.19%, PC2: 26.57%), revealing clear separation between sample groups (Figure 4).

[0240] Among all treatments, cross fermentation sample displayed the most distinct clustering, positioned in the upper-right quadrant of the PCA plot. This sample was strongly associated with propanoic acid, acetic acid, and asparagine, indicating a differentiated metabolic composition. These metabolites are commonly linked to advanced microbial activity and fermentation-derived complexity. The unique positioning of cross fermentation sample reflects the impact of its cross-fermentation strategy, which appears to enable a broader or more targeted biochemical transformation compared to other conditions.

[0241] The second stage fermentation clustered nearby, associated with elevated levels of phenylalanine, leucine, and methionine— amino acids known for contributing bitter taste attributes. This suggests that while microbial transformation is progressing, it may also lead to the accumulation of compounds that influence the sensory profile in less desirable ways, depending on the context and intended application. Compared to cross fermentation sample, the metabolic shift in the second stage fermentation appears more limited in scope and complexity.

[0242] Samples from the first stage fermentation and raw material grouped closely, showing minimal separation and reflecting limited biochemical transformation. Their profiles suggest early or incomplete metabolic activity relative to more developed fermentation stages.

[0243] The Fermentation all microbes sample clustered in a separate quadrant, indicating a distinct metabolic trajectory, though not as compositionally differentiated as cross fermentation sample. Its positioning reflects an alternative fermentation outcome, possibly shaped by microbial interactions within the full inoculum.

[0244] Taken together, the PCA highlights how different fermentation approaches result in distinct and informative metabolic profiles, with cross fermentation sample occupying a notably separate space in the multivariate landscape, reflecting a particularly complex and targeted fermentation outcome within the conditions tested.

[0245] Example 7 - Sensory tests

[0246] Conventional plant proteins often exhibit undesirable attributes such as a pronounced beany taste and notable bitterness, rendering them less appealing for widespread use in food applications. Additionally, achieving a consistent umami flavor profile poses challenges when applying to multiple different food forms.

[0247] The process according to the present invention leverages a synergistic interplay of unique enzyme families sourced from fungi / koji, specific yeast strains, and a combination of bacteria / LAB (Lactic Acid Bacteria). This formulation effectively eliminates undesirable characteristics like beany notes and bitterness, while concurrently developing an ideal, multi-layered umami profile. The process according to the present invention incorporates elements of sweetness, sourness, and complexity, resulting in a harmoniously balanced umami experience. Furthermore, the collaborative action of umami (savoriness) and kokumi (richness) enhances the overall organoleptic profile, ensuring a well-rounded sensory experience. The inclusion of LAB and Propionic bacteria introduces complementary sour notes that enrich the flavor profile, enhancing it beyond what can be achieved through singular fermentation processes.

[0248] Process comparisons in relation to sensory feedback

[0249] A panel comprising 2 to 3 professionals with culinary arts and food science expertise was convened for evaluation. Utilizing a scoring system ranging from 1 (Low) to 7 (High), assessments were made, and subsequently, scores were aggregated to derive a total average score without decimal points for ease of interpretation and simplicity.

[0250] Table 7

[0251] (Scale: 1 Poor - 7 Excellent)

[0252] Table 7 demonstrates the sensory evaluation of different process criteria in order to understand the nuances from synergistic cross-fermentation in V49 (produced by the process of the invention) and other variants. "#1 Koji heat treated," according to Table 7, was produced identically to V49 except that the koji was heat treated with the legumes at the beginning before fermentation started. "#2 Koji and bacteria apart," according to Table 7, was produced identically to V49, except that the fermentation steps was individual for koji and bacterial and thereby not cross-fermented combined, i.e. the first stage was the "koji" fermentation step, followed by heat treatment, then the bacterial fermentation stage. "#3 No yeast," according to Table 7, was produced identically to V49, except it did not include yeast. "#4 Propionic bacteria and LAB apart," according to Table 7, was produced identically to V49, except that the Propionic and LAB were not co-fermenting throughout but added at different stages.

[0253] Table 8

[0254] (Scale: 1 Poor - 7 Excellent)

[0255] Table 8 demonstrates the sensory evaluation of V49 (produced according to the invention) compared to different umami-rich products commonly available commercially or in retail.

[0256] Table 9

[0257] (Scale: 1 Poor - 7 Excellent)

[0258] Table 9 demonstrates the sensory evaluation V49 (produced according to the invention) compared to different plant protein sources products commonly available commercially or in retail.

[0259] Table 10

[0260] (Scale: 1 Poor - 7 Excellent)

[0261] Table 10 shows a sensory assessment of V49 (produced according to the invention) in relation to both process variations and comparisons to umami flavors and plant-based protein products currently available in the market distinctly showcase the distinctive sensory characteristics and benefits of this synergistic cross-fermentation. This notably includes the elimination of undesirable off-flavors or bitterness and the enhancement of desirable umami levels, all achieved while maintaining low salt / sodium levels. Blind Product application sensory test

[0262] Additionally, an application-blind study was conducted to assess the sensory impact of the fermented ingredients in various food applications. A panel comprising four professionals trained in R&D, Culinary, and Food Science underwent a blind tasting of one distinct food form:

[0263] The panel tasted Mushroom risotto (vegetarian) prepared with raw material and different fermented ingredients (cross fermentation, only second submerged fermentation stage, inactive step between two submerged fermentation stages). In dishes featuring the ingredients, they were incorporated at a concentration of 3% of the total food. Mean scores for sensory intensity and liking attributes of four different fermentation samples evaluated in risotto with mushroom broth are list on the table 11

[0264] Table 11 Mean scores for sensory intensity and liking attributes of four different fermentation samples evaluated in risotto with mushroom broth

[0265] In total, 4 samples include 215 (Cross fermentation), 589 (Raw material), 401 (Inactive step between two submerged fermentation stages), and 745 (Only second submerged fermentation stage). Intensity attributes (e.g., saltiness, umaminess, creaminess, bitterness, thickness, continuity, and off-aroma) were rated by consumers on a structured scale from "not at all" to "very strong." Liking attributes (aroma liking and taste liking) were rated on a hedonic scale ranging from "extremely dislike" to "extremely like." Higher scores represent stronger perception or higher liking, depending on the attribute.

[0266] A Principal Component Analysis (PCA) (Figure 5) was conducted based on sensory data collected from a risotto with mushroom broth application to evaluate the performance of different fermentation samples. The sensory attributes included saltiness, umaminess, creaminess, bitterness, and koku-related descriptors: thickness, continuity, and overall liking of aroma, taste, and off- aroma.

[0267] The first two principal components (PCI and PC2) explained the majority of the variance in the data, capturing the main sensory contrasts among the samples.

[0268] Cross fermentation sample (215) was clearly separated in the positive direction, aligning strongly with high saltiness, umaminess, and creaminess, as well as with koku-related sensations such as thickness and continuity. This sample also corresponded to high consumer liking scores in terms of taste and aroma, and low off-aroma, positioning it as the most favorable formulation within the PCA space. Inactive step between two submerged fermentation stages sample (401) clustered near second fermentation stage sample (745), showing moderate association with creaminess and aroma continuity. Despite a partial development of positive sensory attributes, sample 401 did not reach the level of consumer satisfaction or flavor integration observed in cross fermentation sample, suggesting it represents an intermediate stage requiring further fermentation.

[0269] Raw material sample (589), corresponding to the unfermented raw material, was driven by high bitterness and low creaminess and umaminess. This positioning reinforced the role of fermentation in modulating off-flavors and enhancing complexity.

[0270] In summary, PCA confirmed that cross fermentation sample (215) provides the most desirable flavor profile in risotto with mushroom broth, effectively integrating umami, creaminess, saltiness, and koku. The analysis supports the use of a cross-fermentation strategy to achieve such outcomes, highlighting its potential in the development of high-quality savory applications.

[0271] Furthermore sensory feedback was captured as such. Substantial flavor enhancement, increase of depth and umami and heighten overall appeal in the dishes featuring the cross fermented ingredient.

[0272] Example 8 - Multifunctional ingredient attributes Cross fermentation process shapes multifunctional ingredient attributes, which differs from raw ingredient, inactivation between two submerged fermentation stages sample, second submerged fermentation stage sample.

[0273] Each prepared in a water-based formulation containing 20% w / w of the ingredient. Panellists evaluated key sensory attributes, including umami, creaminess, viscosity, bitterness, slipperiness, graininess, and off-flavor, covering both desirable and undesirable qualities relevant to multifunctional ingredient development. The mean sensory attribute intensity score for the four different fermentation samples is listed on the Table 12

[0274] Table 12 Mean sensory attribute intensity scores for the four different fermentation samples

[0275] In total, 4 samples include 147 - Cross fermentation sample, 354 - Raw material sample, 910 - Inactivation between two submerged fermentation stages sample, 842 - Second submerged fermentation stage sample. Values represent average consumer ratings on an intensity scale (e.g., from "not at all" to "very strong") for each sensory attribute, including umami, creaminess, viscosity, bitterness, slipperiness, graininess, and off-flavor. Higher scores indicate stronger perception of the attribute, regardless of whether it is considered desirable or undesirable.

[0276] Following the sensory evaluation, a Principal Component Analysis (PCA) was performed to interpret the data and visualize the main sensory contrasts among the samples (Figure 6). Sample 147 (Cross fermentation) was clearly separated, showing strong associations with umami, creaminess, and viscosity, and minimal off-flavors. This sample also aligned with higher consumer liking scores.

[0277] Sample 354 (Raw material) shows viscosity but lacking developed sensory complexity, reinforcing the role of fermentation in enhancing key attributes.

[0278] Sample 910, produced with inactivation between fermentation stages, was located negatively near bitterness and slipperiness, likely due to incomplete or disrupted fermentation.

[0279] Sample 842, representing a second fermentation stage, was linked to graininess and off-flavor, highlighting potential issues from over-processing or microbial imbalance.

[0280] In summary, the sensory and statistical analyses confirmed that Sample 147 achieved the most desirable sensory profile, thanks to a carefully controlled crossfermentation process. This method enhances flavor and texture while reducing undesirable traits, supporting its potential as a high-performance multifunctional ingredient in savory applications.

[0281] Example 9 - Toxins and Microbial Safety

[0282] The final food product or feed ingredient of the process according to the invention resulted in a microbial-safe end product based on established criteria are summarized in Table 13 , including: sum of all positive aflatoxins (Bl, B2, Gl, G2) not calculable ug / kg, deoxynivalenol (vomitoxin) <20 ug / kg, sum ofT-2 toxin andHT-2 toxin not calculable ug / kg, ochratoxin A. < 0.5 ug / kg, Enterobacteriaceae not calculable, i.e. < 10 CFU / g, moulds < 100 cfu / g and yeast <100 cfu / g, aerobic plate count (APC) <10,000 cfu / g, Listeria monocytogenes not detected / 25g.

[0283] Table 13

[0284] Visual and sensory observations have been conducted during R&D as indicator to microbial safety. On several occasions, observations that would strongly indicate an unsafe microbial product are the visible formation of molds and noticeable off- flavors. Comparisons test of non-cross fermented variants was tested in comparisons to a cross-fermented and notably, discoveries were made in variations without the synergistic effect witnessed:

[0285] • Without cross-fermentation I live fungi and only bacterial culture: resulted in visible pink mold on the surface after the first stage, with some off aroma, notes that become more pronounced after second stage SMF- fermentation.

[0286] • Fungi fermented first with a subsequent fermentation of cultures and vice versa also resulted in a pink mold but at a less degree than the bacterial only. Some umami aroma but noticeably off / unidentifiable aromas

[0287] • All variants of non-cross fermented versions resulted in 6% lower pH indicating less optimal conditions for vitamin B12 generation and a lower umami aroma score.

[0288] Example 10 - Antinutrients

[0289] The use of a combination of Propionibacterium spp., Lactobacillus spp., Saccharomyces spp. and Aspergillus spp (e.g. koji) in a cross-fermentation process appear to have a synergistic effect in reducing the content of phytic acid and accelerating the breakdown process of phytic acid. The breakdown of phytic acid is beneficial as it can enhance the nutritional quality of fermented products. In this process, the content of phytic acid results in a 10- 20% lower phytic acid content compared to non-cross-fermentation processes.

[0290] Example 11 - Protein functionality

[0291] To evaluate the functional properties of the fermented protein obtained by the process according to the invention ("Fermented protein") across various applications, the inventors conducted a comparative study against common plant protein sources including Faba bean protein, Pea protein, and Rice protein. The examination focused on three key aspects, 1) Fat and water binding / emulsification ability, 2) Thermal stability of protein and 3) Solubility effect of protein (room temperature, 25°C) in water.

[0292] 1 ) Fat and water binding / emulsification ability

[0293] Test and results was conducted as such.

[0294] Table 14A (High Oil / Low Water: 20% protein + 30% water + 50% oil)

[0295] (Scale: 1 Poor - 7 Excellent)

[0296] Table 14B (Low Oil / High Water: 20% protein + 55% water + 25% oil)

[0297] (Scale: 1 Poor - 7 Excellent)

[0298] Table 14A) Results:

[0299] Fermented protein: Quick emulsification, thick, short textured, per general favored property. All solids diluted / in suspension.

[0300] Faba bean: Quick emulsification, less viscous than fermented protein, but long thick texture generally not a preferred property. All solids diluted / in suspension.

[0301] Pea protein : Thickening, but not emulsification per see, visible granulates and some water oil separation.

[0302] Rice protein: Thickening, but then separation with significant oil separated from the water / solids mass.

[0303] Table 14B) Results:

[0304] Fermented protein: Quick emulsification, some thickening but much less viscous.

[0305] Faba bean: Quick emulsification, some thickening, some stretchy / gelatinized effect (not a preferred property).

[0306] Pea protein : Thickening and more viscous than Fermented and Faba protein. Granulates visible and present in mouthfeel.

[0307] Rice protein: Thickening, but quick separation and oil seeped out.

[0308] 2) Thermal stability of protein (20% protein and 80% water)

[0309] Table 15

[0310] (Scale: 1 Poor - 7 Excellent)

[0311] The following criteria was applied in the assessment (scoring) of dissolvability, clumping, viscosity impact and suspension over time (20 min) according to Table 15.

[0312] Dissolvability: determine how quickly and well the protein can go into a solution to create a homogenous material.

[0313] Clumping: determine if particles form and clump under just light agitation (Hand stirring).

[0314] Viscosity impact: determine how much more viscous does the product get after heating.

[0315] Suspension : determine how well does the material stay in solution 20 min after heating.

[0316] 20% protein and 80% water was brought to the boiling point under light agitation and the following was observed (Table 15): Fermented protein (of V49): Solids quickly dissolved; some formation of minor clumps formed in the beginning, but by the end heating it was fully dissolved. The material was still in suspension / solution 20 minutes after heating

[0317] Faba bean: The material clumped and thickened more than other proteins, some clumps were noticeable. The material was not in full suspension / solution 20 minutes after heating

[0318] Pea protein : Significant thickening to paste and more viscous than any other compared proteins. Material stayed in suspension 20 minutes after heating

[0319] Rice protein: Solids quickly dissolved, and no clumps were formed. The material was not in full suspension / solution 20 minutes after heating, and solids fell to the bottom.

[0320] 3) Solubility effect of protein (room temperature, 25°C, 20% protein and 80% water mixed under light agitation) in water

[0321] Table 16

[0322] (Scale: 1 Poor - 7 Excellent) The following criteria was applied in the assessment (scoring) of dissolvability, clumping, viscosity impact and suspension over time (20 min) according to Table 16.

[0323] Dissolvability: determine how quickly and well the protein can go into a solution to create a homogenous material.

[0324] Clumping: determine if particles form and clump under just light agitation (Hand stirring).

[0325] Viscosity impact: determine how much more viscous does the product get after heating.

[0326] Suspension : determine how well does the material stay in solution 20 min after heating.

[0327] 20% protein and 80% water was mixed under light agitation and the following was observed (Table 16):

[0328] Fermented protein: Clumps were formed, but within 10 minutes, everything was dissolved, and the material stayed in suspension 20 minutes after.

[0329] Faba bean: Significant clumps formed and did not dissolve within 20 minutes

[0330] Pea protein : Significant thickening to paste and more viscous than any other compared proteins. Noticeable granulates were visible (Not fully dissolved but not clumpy). Material stayed in suspension 20 minutes after. Rice protein: Solids dissolved, and no clumps were formed. The material was not in full suspension / solution 20 minutes after, as solids and fallen to the bottom. References

[0331] Cubillos F. et al., Yeast, "Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers", Vol 36, Issue 6, Pages 383-398, June 2019.

[0332] Clark A. J. et al., LTW, Food Science and Technology, "Shiitake mycelium fermentation improves digestibility, nutritional value, flavor and functionality of plant proteins", Vol 156, 2022.

[0333] Dank A. et al., LTW, Food Science and Technology, "The cross-over fermentation concept and its application in a novel food product: The dairy miso case study", Vol 142, May 2021.

[0334] Deptula P. et al., Front. Microbiol., "Food-like growth conditions support production of active vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the lower ligand base, or cobalt", Vol 8, 8 March 2017.

[0335] Liu D. et al., American Society for Microbiolody, "The Fungal Microbiome Is an Important Component of Vineyard Ecosystems and Correlates with Regional Distinctiveness of Wine", Vol 5, Issue 4, July / August 2020.

[0336] Rees Tom, Euromonitor International, "Plant-Based Foods Face Key Challenges", 3 May 2023.

[0337] Wilson Caleb, Food Business News, "Plant-based meat market at a crossroads amid declining sales", 22 August 2023.

[0338] Smart Protein Project EU, "Evolving appetites: an in-depth look at European attitudes towards plant-based eating", November 2023.

[0339] Xiang, Huan, et al. Food Science and Human Wellness, "Fermentation-enabled wellness foods: A fresh perspective." Vol 8, Issue 3, September 2019. Xia, Yujie, et al., Journal of Texture Studies, "Effects of food components and processing parameters on plant - based meat texture formation and evaluation methods.", Vol 54, Issue 3, June 2023. CN1935009B

[0340] US20200245640A1

[0341] US8153174B2

[0342] WO2023126389

Claims

Claims1. A process for producing a fermented plant-based food product or feed ingredient, the process comprising the steps of: a) mixing legume, whole grain bran and / or seaweed and water, b) heating and subsequently cooling of the mixture resulting from step a), c) inoculating the material resulting from step b) with cl) whole grain solid-state fermented (SSF) in the presence of one or more Aspergillus spp. strains, c2) and one or more Propionibacterium spp. strains, d) first submerged fermenting (SMF) the mixture from step c) in a closed system without stirring, e) inoculating the material resulting from step d) with el) one or more Lactobacillus spp. strains and e2) one or more Saccharomyces spp. strains, f) second submerged fermenting (SMF) the mixture from step e) in a closed system with stirring, g) heating and subsequently cooling of the mixture resulting from step g), h) drying the mixture resulting from step f), thereby providing a fermented food product or feed ingredient.

2. The process according to claim 1, wherein the amounts of the mixing ingredients in step a) are as follows: 10-40% (w / w) of legume, 1-5% (w / w) whole grain bran and / or seaweed and 40-70% water.

3. The process according to any of claims 1-2, wherein the legume in step a) is in the form of legume and is selected from a group consisting of faba bean, pea, soy bean, lupine, black beans, navy beans, chickpeas, lentils or combinations thereof.

4. The process according to any of claims 1-3, wherein the whole grain bran in step a) is selected from a group consisting of rice, oat, wheat, rye or combinations thereof.

5. The process according to any of claims 1-4, wherein the whole grain in step cl) is selected from barley, rice, brown rice, quinoa, oat, farro, spelt, buckwheat or combinations thereof.

6. The process according to any of claims 1-5, wherein the one or more Aspergillus spp. strains in step cl) is / are selected from a group consisting of Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii, Aspergillus luchuensis, Aspergillus sojae, Aspergillus luchuensis awamori, Aspergillus tamariior or combinations thereof.

7. The process according to any of claims 1-6, wherein the one or more Propionibacterium spp. strains in step c2) is / are selected from the group consisting of P. freudenreichii, P. thoenii, P. jensenii, and P. acidipropionici or combinations thereof8. The process according to any of claims 1-7, wherein the one or more Propionibacterium spp. strains in step c2) is / are mixed with the mixture resulting from step cl) to a 1-10 log cfu / g.

9. The process according to any of claims 1-8, wherein the fermentation in step d) is carried out for 8-30 hours at 25-34°C with a VVM of 0-0.5 and at a pH of 4.5-6.

10. The process according to any of claims 1-9, wherein 0.5-5 log cfu / g of the one or more Lactobacillus spp. strains in step el) and 0.3-4% (w / w) of the one or more Saccharomyces spp. strains in step e2) are mixed with the mixture resulting from step d).

11. The process according to any of claims 1-10, wherein the one or more Lactobacillus spp. strains in step el) is / are selected from the group consisting of Lactobacillus easel, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus easel, Lactobacillus brevis, Lactobacillus plantarum or combinations thereof.

12. The process according to any of claims 1-11, wherein the one or more Saccharomyces spp. strains in step e2) is / are selected from the group consistingof Saccharomyces pastorianus, Saccharomyces cerevisiae, Saccharomyces bayanus or combinations thereof.

13. The process according to any of claims 1-12, wherein the fermentation in step f) is carried out for 8-24 hours at 25-34°C with a VVM of 0-0.5 and at a pH of 4.5-6.

14. The process according to any of claims 1-13, wherein the following SST pretreatment steps i) and ii) are carried out before the mixing step a): i) inoculating koji mold spores collected from one or more Aspergillus species onto steamed grains like whole grain (e.g. barley, rice, brown rice, quinoa, oats, farro, spelt, buckwheat), ii) drying of the mixture resulting from step i).

15. A fermented plant-based food product or feed ingredient produced by a process according to any of claims 1-14, wherein the fermented plant-based food product or feed ingredient is having one or more of the following characteristics:(a) NaCI concentration lower than 2% w / w,(b) glutamate content between 100 and 3500mg / 100g dw,(c) vitamin B12 content of more than 5pg / 100g dw,(d) iron content between 40 and 120mg / 100g dw,(e) protein content between 20 and 60 g / lOOg dw,(f) PDCASS (Protein Digestible Corrected Amino Acid Score) >75%,(g) phytic acid reduction of > 10%,(h) improved sensory profile and overall liking via enhanced flavor (umami, saltness) and texture (creaminess, slipperiness and smoothness) while reducing undesirable traits (off-flavor, bitterness and graininess).

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