Preparation method of lactic acid bacteria culture, product of lactic acid bacteria culture and culture medium for lactic acid bacteria culture

Abstract

The invention relates to a microbial starter. More specifically, a method of preparing a microbial culture, such as a lactic acid bacteria (LAB) starter, in which at least one microbial strain, such as lactic acid bacteria, and at least one non-protein binding heme, are inoculated in a culture medium.

Description

Technical Field

[0001] The present invention relates to the field of microbial starter cultures. More specifically, the present invention provides a method for preparing a microbial starter culture under aeration conditions. The microbial starter culture can be a lactic acid bacteria (LAB) starter culture, wherein the lactic acid bacteria are inoculated in a culture medium, and wherein the culture medium contains at least one non-protein-bound heme. This novel method uses vegetarian regulatory compliance raw materials. Therefore, the starter culture obtained by this novel method can be used to manufacture vegetarian foods, feed products and medicines. Technical Background

[0002] Microbial cultures are widely used in the manufacture of fermented products (including most dairy products, such as cheese, yogurt and butter) in the food, feed and pharmaceutical industries, and in the manufacture of meat, baked goods, wine or vegetable products. In addition, microbial cultures are also used to produce proteins (including enzymes) and various useful compounds. Such microbial cultures are commonly referred to as starters, and are produced in industrial breeding factories and distributed to fermentation industries, such as dairies, where starters are used for their production processes. Especially, lactic acid bacteria cultures are widely used as starters.

[0003] The production of lactic acid bacteria (LAB) starter cultures involves inoculating LAB cells in a specific fermentation medium, and under appropriate fermentation conditions, an appropriate number of cells are propagated. In an industrial setting, a lot of effort is put into obtaining a high concentration of propagating cells near the end of the fermentation process. Fermentation conditions and fermentation medium must support the growth of cells to obtain the desired high biomass yield.

[0004] Current methods for producing lactic acid bacteria starters, such as Lactococcus lactis starters, utilize non-vegetarian compliant sources as raw materials in the fermentation medium. The non-vegetarian compliant source is used as an exogenous source. The exogenous source may be a heme source, and it is added to support the respiratory process of the lactic acid bacteria. Due to the use of a non-vegetarian compliant heme source, such starters obtained by known methods cannot be used for vegetarian foods, feeds and pharmaceuticals. Therefore, there is a need in the art to develop a respiratory process for producing microbial starters, such as lactic acid bacteria, with a yield similar to that of processes known in the art, and wherein the process utilizes a vegetarian compliant heme source.

[0005] As disclosed in WO2021 / 116311A1, yeast cells have been implemented as a vegetarian compliant heme source. However, the production, processing and purification of yeast cells are cumbersome, which can lead to increased costs. In addition to LAB biomass, yeast cells also increase dry matter. Using yeast cell-based materials in the form of yeast extracts will add additional processing steps to the preparation of materials containing non-protein-bound heme.

[0006] Microorganisms can be used to express heme, for example as shown in EP 3567109 entitled extracellular heme production method using metabolically engineered microorganisms. However, it is not clear whether such heme can be maintained in cell culture. SUMMARY OF THE INVENTION

[0008] The problem to be solved by the present invention is to provide a microbial culture (eg a lactic acid bacteria culture) which is suitable for the production of vegetarian food, feed products and pharmaceuticals.

[0009] Therefore, a first aspect of the present invention relates to a method for obtaining a microbial culture, the method comprising the following steps:

[0010] (i) culturing at least one microbial strain in a culture medium under aeration conditions and obtaining a fermentation product,

[0011] (ii) harvesting the at least one microbial strain from the fermentation to obtain a microbial culture,

[0012] The culture medium comprises at least one non-protein-bound heme.

[0013] In a second aspect, the invention relates to a culture obtainable by the method of the invention.

[0014] In a third aspect, the invention relates to a culture comprising at least one non-protein-bound heme.

[0015] A fourth aspect of the present invention relates to a culture medium comprising at least one non-protein-bound heme.

[0016] A fifth aspect of the present invention relates to a method for preparing a food, feed product, pharmaceutical, dairy flavor and cheese flavor product, the method comprising adding an effective amount of the culture of the present invention to the food, feed product or pharmaceutical starting material and maintaining the inoculated culture under conditions where at least one microbial strain is metabolically active.

[0017] A sixth aspect of the present invention relates to a fermented food, feed or pharmaceutical product obtainable by the method of the present invention.

[0018] A seventh aspect of the present invention relates to the use of at least one non-protein-bound heme in a fermentation method and / or a fermentation process.

[0019] An eighth aspect of the present invention relates to a food, a feed product, a pharmaceutical product, a dairy flavor or a cheese flavor product, comprising the culture according to the second aspect or the third aspect.

[0020] Detailed disclosure of the invention

[0021] The inventors have developed a method for obtaining a microbial culture, such as a starter culture of a microbial strain (e.g., lactic acid bacteria), wherein non-protein-bound heme is used as a vegetarian compliance alternative heme source rather than a non-vegetarian compliance heme source. The use of non-protein-bound heme as an exogenous heme source surprisingly showed support for the respiration of microbial strains (e.g., lactic acid bacteria). The purified non-protein-bound heme is a vegetarian compliance raw material. The yield provided by the method is comparable to methods known in the art.

[0022] Before discussing detailed embodiments of the present invention, further definitions of selected terms used herein are provided.

[0023] As used herein, the term "non-protein-bound heme" refers to free heme that is not bound to a protein containing a heme prosthetic group.

[0024] As used herein, the term "fermentation" refers to a process of multiplying or culturing microbial cells under aerobic or anaerobic conditions.

[0025] The term "starter culture" refers to a preparation comprising microbial cells intended for inoculation of a medium to be fermented.

[0026] In this context, the term "yield" refers to the amount of biomass produced in a given volume of fermentation. Yield can be measured in a number of ways: 1) as the optical density at 600 nm (OD 600 ) per unit volume of biomass (minus background); 2) "acidification activity" or acidification power of 4.8-5.2 according to the Pearce test in kg of F-DVS culture at the end of fermentation; 3) by the packed cell volume (PCV) test; or 4) cell counts.

[0027] The term "F-DVS" refers to a so-called Frozen Direct Vat Set culture, as described in the Examples.

[0028] The legal framework in Europe regarding vegetarian claims is currently under revision and there are no harmonized rules yet. All claims under European food legislation, vegan and vegetarian claims are any information or representation not mandatory under EU or national legislation, including any form of pictorial representation, graphic representation or symbolic representation, which illustrates, indicates or implies that a food has specific characteristics (Neli Sochirca (2018), EFFL, 6, p. 514). Therefore, in this context, the term "vegetarian compliant heme source" refers to a heme source that is not obtained or derived from animals and / or multicellular organisms. In contrast, the term "non-vegetarian compliant heme source" refers to a heme source that is obtained or derived from animals and / or multicellular structures.

[0029] In an embodiment of the present invention, one or more microbial strains are microbial strains that are not capable of respiratory growth without supplementation of components / alternative components of the respiratory chain. It should be understood that supplementation of components / alternative components of the respiratory chain can be supplementation of an exogenous heme source.

[0030] At least one microbial strain may be selected from the group consisting of: Lactococcus, Streptococcus, Lactobacillus (now called Ligilactobacillus), Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquidilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus (such as Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, Bifidobacterium, Brevibacterium, Propionibacterium, and combinations thereof.Most genera in this group are "lactic acid bacteria", however, one industrially important genus is Bifidobacterium, which, although phylogenetically unrelated, is sometimes included in the group of lactic acid bacteria because lactic acid is one of the major fermentation end products. The list also includes other industrially important fermentation agents that are not included in the group of lactic acid bacteria but belong to the genera Brevibacterium and Propionibacterium.

[0031] As used herein, the term "lactic acid bacteria" (LAB) refers to Gram-positive, microaerophilic or anaerobic bacteria that ferment sugars and produce acids, including lactic acid (as the predominantly produced acid) and acetic acid. The most industrially useful lactic acid bacteria are found in the following genera: Lactococcus, Streptococcus, Lactobacillus (now called Lactobacillus), Holzapfel, Lactobacillus amyloliquefaciens, Lactobacillus bee, Lactobacillus associative, Lactobacillus stonewall, Lactobacillus agriculturae, Lactobacillus schleifer, Lactobacillus putrefaciens, Lactobacillus casei, Lactobacillus extensive, Draglio, Lactobacillus liquidus, Lactobacillus united, Lactobacillus plantarum, Lactobacillus furfur, Lactobacillus oligovorus, Lactobacillus mucus, Lactobacillus fruit, Lactobacillus aceticus, Lactobacillus bee, Lactobacillus promotility, Lactobacillus subtilis and Lactobacillus lentus (as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc, Oenococcus, Weissella, Pediococcus and Enterococcus. As mentioned above, another industrially important genus is Bifidobacterium, which, although phylogenetically unrelated, is sometimes included in the group of lactic acid bacteria because lactic acid is one of the major fermentation end-products.

[0032] Thus, in one embodiment, at least one microbial strain is a lactic acid bacterium selected from the group consisting of Lactococcus, Streptococcus, Lactobacillus (now called Lactobacillus united), Holzapfel, Lactobacillus amyloliquefaciens, Lactobacillus bee, Lactobacillus associative, Lactobacillus stonewall, Lactobacillus agriculturae, Lactobacillus schleiferi, Lactobacillus putrefaciens, Lactobacillus casei, Lactobacillus extensive, Draglio, Lactobacillus liquidus, Lactobacillus plantarum, Lactobacillus furfur, Lactobacillus oligovorus, Lactobacillus mucus, Lactobacillus fruiti, Lactobacillus aceticus, Lactobacillus bee, Lactobacillus promotility, Lactobacillus subtilis and Lactobacillus lentus (as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI: 10.1007 / 0000-0100). 10.1099 / ijsem.0.004107), Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, Bifidobacterium, and combinations thereof.

[0033] Commonly used LAB starter lactic acid bacteria strains are generally divided into mesophilic organisms having an optimal growth temperature of about 30°C and thermophilic organisms having an optimal growth temperature ranging from about 40°C to about 45°C.

[0034] It should be understood that the Lactobacillus taxonomy has been updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020, and the lactobacilli important to the present invention are summarized as follows:

[0035]

[0036] Typical organisms belonging to the mesophilic group include Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostocmesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei (Lactococcus casei), and Lactobacillus paracasei subsp. casei (Lactococcus paracasei and Lactobacillus paracasei subsp. firmus). Species of thermophilic lactic acid bacteria include, for example, Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus.

[0037] Since the amount and therefore the concentration of non-protein-bound heme, lactic acid bacteria, non-protein-bound heme or any other nutrient in the culture medium may change over time (e.g. due to incorporation into microbial cells), it is necessary to indicate the specific time point at which the concentration of non-protein-bound heme must be measured or determined. Therefore, when used in relation to the concentration of non-protein-bound heme, lactic acid bacteria, non-protein-bound heme or any other nutrient in the culture medium, the term "initially" or "before fermentation" (also used interchangeably herein) refers to the concentration of non-protein-bound heme, lactic acid bacteria, non-protein-bound heme or any other nutrient present in the culture medium immediately before the microbial cells to be cultured are added to the culture medium.

[0038] However, non-protein bound heme can also be added at any time before harvest for the entire fermentation process. The addition of non-protein bound heme can be done batchwise or continuously. Therefore, an important measurement is the "total amount added" during the entire fermentation process.

[0039] An important application of the starter according to the invention is as so-called probiotics. In the present context, the term "probiotics" is to be understood as microbial cultures which, when ingested in the form of living cells by humans or animals, confer improved health conditions (e.g. by inhibiting harmful microorganisms in the gastrointestinal tract, by enhancing the immune system or by promoting the digestion of nutrients). A typical example of such a probiotic active product is "sweet acidophilus milk".

[0040] Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0041] Unless otherwise indicated by the context, the embodiments, preferences and options of a given aspect, feature or parameter of the present invention should be considered as having been disclosed in conjunction with any and all embodiments, preferences and options of all other aspects, embodiments, features and parameters of the present invention. For example, the embodiments relevant to the lactic acid bacteria culture that can be obtained by the method of the present invention are equally applicable to lactic acid bacteria starter. In addition, the embodiments described about the method of the present invention may be relevant to the product of the present invention, and vice versa.

[0042] Embodiments of the invention are described below by way of example only.

[0043] One aspect of the present invention relates to a method for obtaining a microbial culture, the method comprising the following steps:

[0044] (i) culturing at least one microbial strain in a culture medium under aeration conditions and obtaining a fermentation product,

[0045] (ii) harvesting the microbial strain from the fermentation to obtain a microbial culture,

[0046] The culture medium comprises at least one non-protein-bound heme.

[0047] In an embodiment, the present invention relates to a method for obtaining a lactic acid bacteria culture, the method comprising the following steps:

[0048] (i) culturing at least one lactic acid bacteria culture in a culture medium under aeration conditions and obtaining a fermentation product,

[0049] (ii) harvesting the microbial strain from the fermentation to obtain a microbial culture,

[0050] The culture medium comprises at least one non-protein-bound heme.

[0051] In one embodiment, the method of the present invention may further comprise the following steps:

[0052] (iii) concentrating the microbial culture to obtain a concentrated microbial culture.

[0053] In one embodiment, the method of the present invention may further comprise the following steps:

[0054] (iii) concentrating the lactic acid bacteria culture to obtain concentrated lactic acid bacteria.

[0055] The concentration may be carried out using methods known in the art, such as, but not limited to, centrifugation or ultrafiltration. In order to obtain an increased number of microorganisms (e.g., lactic acid bacteria) in the concentrate obtained in step (iii), it may be considered that the concentration factor in step (iv) is in the range of 2 to 20, such as in the range of 6-19, such as in the range of 7-18, such as 8-17, such as 9-16, such as 10-15, such as 11-14, such as 12-13, such as 2-4, such as 3-6.

[0056] Commercial starter cultures are often distributed as frozen cultures. At the low temperatures at which such frozen cultures are usually maintained, most metabolic activity in the cells is stopped, and the cells can be maintained in this suspended but living state for a long time.

[0057] Concentrated frozen cultures are of great commercial interest because they can be directly inoculated into production vessels. By using such concentrated frozen cultures, the end user avoids the otherwise necessary time-consuming intermediate fermentation step in which the starter is expanded, and the end user also significantly reduces the risk of contamination. Such concentrated cultures may be referred to as DVS - Direct Vase Starter TM Culture.

[0058] As an alternative to concentrated frozen cultures, concentrated freeze-dried direct-pitch starter cultures can be prepared TM Culture, FD-DVS TM Such cultures have the additional advantage that they can be transported without refrigeration.

[0059] Therefore, in an embodiment, the method of the present invention may further comprise the following steps:

[0060] (iv) freezing the microbial bacterial culture of step (ii) or the concentrated microbial culture of step (iii) to obtain a frozen microbial culture.

[0061] Therefore, in an embodiment, the method of the present invention may further comprise the following steps:

[0062] (iv) freezing the lactic acid bacteria culture of step (ii) or the concentrated lactic acid bacteria of step (iii) to obtain a frozen lactic acid bacteria culture.

[0063] In order to remove liquid from the frozen microbial bacterial culture, the method of the present invention may further comprise the following steps:

[0064] (v) sublimating water from the frozen microorganism culture to obtain a dried microorganism culture.

[0065] In order to remove liquid from the frozen lactic acid bacteria culture, the method of the present invention may further comprise the following steps:

[0066] (v) sublimating water from the frozen lactic acid bacteria culture to obtain a dried lactic acid bacteria culture.

[0067] Step (v) may be performed by a technique selected from the group consisting of spray drying, spray freezing, vacuum drying, air drying, freeze drying, tray drying and vacuum tray drying.

[0068] In yet another embodiment, the method of the present invention further comprises the following steps:

[0069] (vii) packaging the frozen microbial culture obtained in step (iv) or the freeze-dried microbial culture obtained in step (v).

[0070] It is understood that the method of the present invention further comprises the following steps:

[0071] (vii) packaging the frozen lactic acid bacteria culture obtained in step (iv) or the dried lactic acid bacteria culture obtained in step (v).

[0072] Destructive effects of freezing and thawing on the viability of living cells are frequently observed. Generally, they are attributed to cell dehydration and the formation of ice crystals in the cytosol during freezing.

[0073] However, a number of cryoprotectants have been found that are capable of ensuring that freezing occurs in a controlled and minimally damaging manner (eg by ensuring that ice crystallization in the cytosol is prevented or minimised during freezing).

[0074] Preferably, at least one cryoprotectant is added to the harvested microbial culture or the harvested lactic acid bacteria culture obtained in step (ii) or the concentrated microbial culture or the concentrated lactic acid bacteria culture obtained in step (iii).

[0075] Preferably, the cryoprotectant is selected from the group consisting of one or more compounds involved in nucleic acid biosynthesis or one or more derivatives of any such compounds. Examples of preferred cryoprotectants suitable for addition to harvested microorganisms correspond substantially to preferred non-protein-bound heme as described herein. In the earlier submitted patent application for application number PCT / DK2004 / 000477, it is described to add such cryoprotectants to harvested microorganisms. The preferred cryoprotectants described in PCT / DK2004 / 000477 are also preferred cryoprotectants of the present invention. The complete description of PCT / DK2004 / 000477 is incorporated herein by reference. In another preferred embodiment of the present invention, one or more cryoprotectants are selected from the group of nucleoside monophosphates. In a preferred embodiment, at least one or the only cryoprotectant is IMP. Carbohydrate or protein cryoprotectants are generally not described as increasing the metabolic activity of thawed or reconstructed cultures. The cryoprotectants of the present invention, when inoculated into a culture medium to be fermented, processed or converted, can also impart increased metabolic activity (enhancing effect) to the culture in addition to their cryoprotective activity. Therefore, one embodiment of the present invention is a frozen culture or a dried culture, wherein the cryoprotectant is a mixture of one or more than one agent, and in addition to its cryoprotective properties, the cryoprotectant has an enhancing effect. The expression "enhancing effect" is used to describe a situation in which, when the cryoprotectant is inoculated into a culture medium to be fermented or converted, the cryoprotectant imparts an increased metabolic activity (enhancing effect) to the thawed or reconstructed culture. Vitality and metabolic activity are not synonymous concepts. Commercially frozen or dried (e.g., freeze-dried) cultures can maintain their vitality, although they may have lost a large part of their metabolic activity, for example, when stored (even if the storage time is short), the culture may lose its acidogenic (acidifying) activity. Therefore, vitality and enhancing effects must be evaluated by different assays. Vitality is evaluated by vigor assays (e.g., determining colony forming units), while enhancing effects are evaluated by quantifying the relevant metabolic activity of the thawed or reconstructed culture relative to the vitality of the culture.

[0076] The acidification activity assay described below is one example of an assay to quantify the relevant metabolic activity of thawed or reconstituted cultures.

[0077] Although acidogenic activity is exemplified herein, the present invention is intended to include any type of metabolic activity of a stable culture. Thus, the term "metabolic activity" refers to the oxygen removal activity of the culture, its acidogenic activity (i.e., the production of, for example, lactic acid, acetic acid, formic acid, and / or propionic acid), or its activity to produce metabolites, such as the production of aromatic compounds such as acetaldehyde (α-acetolactate, acetoin, diacetyl, and 2,3-butanediol (butanediol)).

[0078] In embodiments of the invention, the frozen culture contains or comprises 0.2% to 20% of a cryoprotectant or mixture of agents (measured as % w / w of the frozen material). However, it is preferred to add a cryoprotectant or mixture of agents in an amount in the range of 0.2% to 15% by weight, more preferably in the range of 0.2% to 10%, more preferably in the range of 0.5% to 7%, and more preferably in the range of 1% to 6%, including a cryoprotectant or mixture of agents in the range of 2% to 5% (measured as % w / w of the frozen material by weight). In a preferred embodiment, the culture comprises about 3% of a cryoprotectant or mixture of agents (measured as % w / w of the frozen material by weight). The preferred amount of about 3% cryoprotectant corresponds to a concentration in the range of 100 mM. It should be appreciated that for each aspect of the embodiments of the present invention, the range can be an increment of the described range.

[0079] Where the culture is a dried culture (e.g., freeze-dried culture), the cryoprotectant or mixture of agents is preferably added in an amount in the range of 0.8% to 60% by weight, or in the range of 0.8% to 55% by weight, or in the range of 1.3% to 40% by weight, or in the range of 3% to 30% by weight, or in the range of 6% to 25% by weight, including in the range of 10% to 24% by weight of the dried culture. In a preferred embodiment, the dried culture (e.g., freeze-dried culture) comprises about 16% of the cryoprotectant or mixture of agents (measured as % w / w of the dried culture).

[0080] In addition, the frozen culture or dried culture may further contain conventional additives, including nutrients such as yeast extract, sugar, antioxidants, inert gases and vitamins, etc. Surfactants of the compound may also be used as additional additives for the culture according to the invention. Other examples of such conventional additives that may be additionally added to the culture according to the invention may be selected from proteins, protein hydrolysates and amino acids. Preferred suitable examples of these additives include additives selected from the group consisting of glutamic acid, lysine, sodium glutamate, sodium caseinate, malt extract, skim milk powder, whey powder, yeast extract, gluten, collagen, gelatin, elastin, keratin and albumin or mixtures thereof.

[0081] More preferably, conventional additives are carbohydrates. Suitable examples of these additives include additives selected from the group consisting of pentoses (e.g., ribose, xylose), hexoses (e.g., fructose, mannose, sorbose), disaccharides (e.g., sucrose, trehalose, melibiose, lactulose), oligosaccharides (e.g., raffinose), oligofructose (e.g., Actilight, Fribroloses), polysaccharides (e.g., maltodextrin, xanthan gum, pectin, alginate, microcrystalline cellulose, dextran, PEG) and sugar alcohols (sorbitol, mannitol and inositol).

[0082] It is currently preferred that the ratio (wt% / wt%) of at least one cryoprotectant to the concentrated microbial culture or concentrated lactic acid bacteria culture is in the range of 1:0.5 to 1:5, such as 1:1 to 1:4 or 1:1. 1 / 2 to 1:3.

[0083] An alternative embodiment of the present invention is a method for preparing a microbial culture with increased yield as described herein, and the method further comprises drying the concentrated microbial culture or the concentrated lactic acid bacteria culture obtained in step (iii) by freeze drying, tray drying, spray drying, spray freezing, vacuum drying, air drying or any drying process suitable for drying bacterial cultures.

[0084] At least one non-protein-bound heme may be present in the culture medium, or at least one non-protein-bound heme may be added to the culture medium before adding at least one microbial strain and / or lactic acid bacteria to the culture medium, or alternatively, at least one non-protein-bound heme may be added immediately after adding at least one microbial strain and / or lactic acid bacteria to the culture medium.

[0085] In one embodiment, at least one non-protein bound heme is a microorganism expressed protein. In a preferred embodiment, the non-protein bound heme is heme expressed according to EP3567109 entitled Methods for producing extracellular heme using metabolically engineered microorganisms.

[0086] In one embodiment, the composition comprising at least one non-protein bound heme has been deactivated. Several deactivation methods can be used to achieve the purpose of deactivating the natural biological catalytic activity, such as pH (alkaline) deactivation, enzyme digestion or heat deactivation. In a preferred embodiment, the deactivation is heat deactivation. Heat deactivation can be carried out by any method known in the art, such as but not limited to autoclaving and / or UHT.

[0087] The inventors surprisingly found that the addition of a heat stable compound enables industrial processing of non-protein bound heme while achieving high growth yields. In one embodiment, the culture medium further comprises a heat stable compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers and non-ionic detergents. The heat stable compound can be selected from the group consisting of: sorbitol, glycerol, propylene glycol, mannitol, xylitol, propanediol, trehalose, sucrose, lactose, maltose, glucose, fructan (fructose homopolysaccharide), dextran, dextran sulfate, gelatin (type A and type B), hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, fucoidan, pentosan polysulfate, keratin sulfate, polyaspartic acid, polyglutamic acid, hydroxyethylcellulose, hydroxypropyl-β-cyclodextrin, glycine, L-arginine hydrochloride, arginine, proline, lysine, histidine, aspartic acid, glutamic acid, acetate, citrate, sodium chloride, phosphate, ascorbate, poly(acrylic acid) randomly modified with n-octylamine and isopropylamine (A8-35), polyethylene glycol (PEG), polyvinyl sulfate, polysorbate 20, polysorbate 80, Triton X-100, Pluronic F68, Pluronic F88, Pluronic F-127, Brij 35 (polyoxyethylene alkyl ether). In a preferred embodiment, the heat stabilizing compound is sorbitol.

[0088] In one embodiment, the non-protein-bound heme is a protein that has no native biological activity.

[0089] In one embodiment, non-protein-bound heme is produced by a microorganism. In one embodiment, non-protein-bound heme is indirectly derived from an organism of the genus Aspergillus (Aspergillus niger) or is directly produced therefrom. In one embodiment, non-protein-bound heme is indirectly derived from an organism of the genus Pichia (Pichia) (e.g., Pichia pastoris) or is directly produced therefrom. In one embodiment, non-protein-bound heme is indirectly derived from an organism of the genus Saccharomyces (Saccharomyces cerevisiae) or is directly produced therefrom. In one embodiment, non-protein-bound heme is indirectly derived from an organism of the genus Escherichia (Escherichia coli) or is directly produced therefrom. In one embodiment, non-protein-bound heme is indirectly derived from an organism of the genus Bacillus (Bacillus) (e.g., a spore-free Bacillus) or is directly produced therefrom.

[0090] At least one non-protein-bound heme is added to the culture medium or is present in the culture medium as a raw material intended for aiding fermentation. The inventors surprisingly found that the non-vegetarian source used in the culture medium can be replaced with at least one non-protein-bound heme without reducing the yield.

[0091] Therefore, one aspect of the present invention relates to the use of at least one non-protein-bound heme in a fermentation method and / or a fermentation process.

[0092] The culture medium may be a complex fermentation medium.

[0093] The complex fermentation medium may be any complex fermentation medium known in the art, however the complex fermentation medium may comprise a compound selected from the group consisting of lactose, nutrients, vitamins tryptone, soy peptone, yeast extract, ascorbic acid, magnesium sulfate, milk and combinations thereof.

[0094] In one embodiment, the non-protein-bound heme is added at a level that allows respiration to be above the natural oxygen consumption level that the cells can support. The non-protein-bound heme stimulates aerobic microbial growth in a dose-dependent manner, such that oxygen consumption, a measure of microbial growth, peaks earlier and at a faster rate than in cultures without the non-protein-bound heme.

[0095] Thus, in one embodiment, oxygen consumption in the fermentation reaches its maximum in less than 12 hours, such as less than 10 hours or less than 8 hours.

[0096] In one embodiment, oxygen consumption in the fermentation reaches 0.04 mol O2 / L / h in less than 10 hours or less than 8 hours.

[0097] Oxygen consumption can be measured using any method known to those skilled in the art.

[0098] In one embodiment, the culture medium in step (i) comprises at least 0.005% w / w of at least one non-protein-bound heme before fermentation (i.e. before the addition of at least one microbial strain), for example 0.008% w / w, 0.01% w / w, 0.014% w / w, 0.03% w / w or 0.5% w / w, for example in the range of 0.008% w / w to 0.014% w / w, 0.01% w / w to 0.03% w / w, 0.01% w / w to 0.5% w / w or 0.005% w / w to 0.5% w / w of at least one non-protein-bound heme.

[0099] In a preferred embodiment, the concentration of non-protein-bound heme in the culture medium of step (i) is 0.008% w / w to 0.014% w / w.

[0100] The concentration of non-protein bound heme in the culture medium is about 0.008% w / w, for example 0.008% w / w, which corresponds to about 9 ppm to 10 ppm heme, and is particularly suitable for UHT sterilization.

[0101] The concentration of non-protein bound heme in the culture medium is about 0.014% w / w, such as 0.014% w / w, equivalent to about 16 ppm to 17 ppm heme, and is particularly suitable for filter sterilization.

[0102] In other embodiments, the culture medium in step (i) comprises at least 0.5% w / w of a microbial inoculum culture (e.g., a lactic acid bacteria inoculum culture) relative to the weight of the culture medium (i.e., before adding at least one microbial strain) before fermentation, e.g., at least 1% w / w, e.g., 1.5% w / w, e.g., 2% w / w, e.g., 2.5% w / w, e.g., 3% w / w, e.g., 3.5% w / w, e.g., 4% w / w, e.g., within the range of 0.5% w / w-4% w / w, e.g., 1% w / w-3.5% w / w, e.g., 1.5% w / w-3% w / w, e.g., comprising 2% w / w-2.5% w / w of a lactic acid bacteria inoculum culture before fermentation. The inoculum culture can be prepared according to the method specified in Example 1.

[0103] In one embodiment, non-protein bound heme is added to achieve a concentration of about 0.1 g / kg fermentate to about 10 g / kg fermentate.

[0104] Surprisingly, by the method of the present invention, it is occasionally possible to obtain a microbial culture (e.g., a lactic acid bacteria culture) that is sufficiently concentrated for the production of F-DVS without the need to concentrate the culture. However, even with the method of the present invention, most cultures require concentration to obtain a commercially valuable starter. Such cultures can preferably be harvested and concentrated by centrifugation or ultrafiltration.

[0105] Furthermore, a preferred embodiment is one in which the cultivation is carried out in a large fermenter containing 5 L to 100.000 L of the culture medium, preferably 300 L to 20.000 L of the culture medium.

[0106] A preferred embodiment is wherein the culturing comprises controlling temperature and / or pH.

[0107] In an embodiment, the culture medium in step (i) and / or step (ii) comprises one or more microbial strains which are microbial strains that are not capable of respiratory growth without supplementation of components / alternative components of the respiratory chain.

[0108] In an embodiment, the culture medium in step (i) and / or step (ii) comprises at least one microbial strain selected from the group consisting of Lactococcus, Streptococcus, Lactobacillus (now called Lactobacillus), Holzapfel, Lactobacillus amyloliquefaciens, Lactobacillus bee, Lactobacillus associative, Lactobacillus stonewall, Lactobacillus agriculturae, Lactobacillus schleiferi, Lactobacillus putrefaciens, Lactobacillus casei, Lactobacillus extensive, Draglio, Lactobacillus liquidus, Lactobacillus united, Lactobacillus plantarum, Lactobacillus furfur, Lactobacillus oligovorus, Lactobacillus mucus, Lactobacillus fruit, Lactobacillus aceticus, Lactobacillus bee, Lactobacillus promotor, Lactobacillus subtilis and Lactobacillus lentus (as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI: 10.1006 / 000400). 10.1099 / ijsem.0.004107), Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, Bifidobacterium, Brevibacterium, Propionibacterium, and combinations thereof.

[0109] In an embodiment, the culture medium in step (i) and / or step (ii) comprises at least one lactic acid bacterium selected from the group consisting of Lactococcus, Streptococcus, Lactobacillus (now called Lactobacillus), Holzapfel, Lactobacillus amyloliquefaciens, Lactobacillus bee, Lactobacillus associative, Lactobacillus stonewall, Lactobacillus agriculturae, Lactobacillus schleiferi, Lactobacillus putrefaciens, Lactobacillus casei, Lactobacillus extensive, Draglio, Lactobacillus liquidus, Lactobacillus united, Lactobacillus plantarum, Lactobacillus furfur, Lactobacillus oligovorus, Lactobacillus mucus, Lactobacillus fruit, Lactobacillus aceticus, Lactobacillus bee, Lactobacillus promotility, Lactobacillus subtilis and Lactobacillus lentus (as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI: 10.1006 / 000400). 10.1099 / ijsem.0.004107), Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, and Bifidobacterium.

[0110] In an embodiment, the culture medium in step (i) and / or step (ii) comprises one or more mesophilic organisms selected from the group comprising: Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis var. diacetyl, Lactobacillus casei subsp. casei (new name Lactobacillus casei), Lactobacillus paracasei subsp. casei (Lactobacillus paracasei subsp. casei and Lactobacillus paracasei subsp. firmus) and Oenococcus oeni.

[0111] In further embodiments, the culture medium in step (i) and / or step (ii) comprises one or more thermophilic organisms having an optimum growth temperature of about 40°C to about 45°C.

[0112] In another embodiment, the culture medium in step (i) and / or step (ii) comprises one or more thermophilic organisms selected from the group comprising: Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

[0113] In an embodiment, the culture medium in step (i) and / or step (ii) is a LD culture comprising one or more organisms selected from the group comprising: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis var. diacetyl and Leuconostoc mesenteroides subsp. cremoris. In the present context, the term "LD-culture" is to be understood as a combination of Lactococcus lactis species and Leuconostoc species.

[0114] It will be appreciated that the culture medium in step (i) and / or step (ii) is an O-culture comprising one or more organisms selected from the group comprising Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. In this context, an "O-culture" is understood to be a culture medium comprising Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. O-cultures are commonly used to prepare cheese without holes (cheddar, Cheshire, feta). This particular culture is available from Chr. Hansen A / S, Denmark, under the name R 604 (Catalog No. 200113).

[0115] In a preferred embodiment, the culture medium in step (i) and / or step (ii) is a culture comprising Lactococcus lactis.

[0116] In order to obtain the highest yield, it may be preferred to perform the harvesting in step (ii) 5 to 24 hours after the start of the culture.

[0117] The method of the present invention may further comprise storing the harvested microbial culture or lactic acid bacteria culture obtained in step (ii), or the concentrated microbial culture or lactic acid bacteria culture obtained in step (iii).

[0118] Due to the high yield of the method, the microbial culture in the fermentation obtained in step (i) may contain a range of 2.0E+10–5.0E+10 active microbial cells / g microbial culture, such as 2.5E+10–4.5E+10, such as 3.0E+10–4.0E+10 active microbial cells / g microbial culture. Similarly, the microbial culture in the fermentation obtained in step (i) may contain a range of 2,0E+10–5,0E+10 total number of microbial cells / g microbial culture, such as 2.5E+10–4.5E+10, such as 3.0E+10–4.0E+10 total number of microbial cells / g microbial culture. As can be seen in Table 2 of the experimental part, the number of active lactic acid bacteria cells and the total number of lactic acid bacteria cells are almost the same, indicating that the lactic acid bacteria culture and lactic acid bacteria starter that can be obtained by the present invention have high vitality.

[0119] Due to the high yield of the method, the lactic acid bacteria culture in the fermentation obtained in step (i) may contain a range of 2.0E+10–5.0E+10 active lactic acid bacteria cells / g lactic acid bacteria culture, such as 2.5E+10–4.5E+10, such as 3.0E+10–4.0E+10 active lactic acid bacteria cells / g lactic acid bacteria culture. Similarly, the lactic acid bacteria culture in the fermentation obtained in step (i) may contain a range of 2,0E+10–5,0E+10 total number of lactic acid bacteria cells / g lactic acid bacteria culture, such as 2.5E+10–4.5E+10, such as 3.0E+10–4.0E+10 total number of lactic acid bacteria cells / g lactic acid bacteria culture. As can be seen in Table 2 of the experimental part, the number of active lactic acid bacteria cells and the total number of lactic acid bacteria cells are almost the same, indicating that the lactic acid bacteria culture and lactic acid bacteria starter that can be obtained by the present invention have high vitality.

[0120] The number of viable cells and / or total cells is determined using flow cytometry, which is a technique known to the skilled person.

[0121] In a preferred embodiment, the increased yield of the harvested microbial strain (e.g., lactic acid bacteria) or the microbial culture of the method (e.g., lactic acid bacteria culture) is increased by at least 1.2 times, preferably at least 1.3 times, more preferably at least 1.4 times, even more preferably at least 1.5 times and most preferably at least 1.6 times compared to an anaerobic process that does not include a heme source process.

[0122] In a second aspect, the present invention relates to a microbial culture, such as a starter culture, obtainable by the method of the first aspect of the present invention. The microbial culture, such as a starter culture, may be provided as a culture concentrate, such as a starter culture concentrate.

[0123] In a third aspect, the present invention relates to a microbial culture, such as a starter culture, comprising at least one non-protein-bound heme.

[0124] A fourth aspect relates to a culture medium comprising at least one non-protein-bound heme.

[0125] In a fifth aspect, the present invention relates to a method for preparing a food, a feed product, a medicine, a dairy flavor and a cheese flavor product, the method comprising adding an effective amount of a culture according to the second aspect or the third aspect to a food, a feed product or a medicine starting material, and maintaining the inoculated culture under conditions where at least one microbial strain is metabolically active.

[0126] Preferably, the food product of the fifth aspect of the invention is selected from the group consisting of dairy-based products, vegetable products, meat products, beverages, juices, wines, bakery products, dairy flavors and cheese flavored products.

[0127] Preferably, the dairy based product is selected from the group consisting of cheese, yoghurt, butter, inoculated sweet milk and liquid fermented dairy products.

[0128] In a sixth aspect, the present invention relates to a fermented food, feed or medicine obtainable by the method of the first aspect.

[0129] A seventh aspect of the present invention relates to the use of at least one non-protein-bound heme in a fermentation method and / or a fermentation process.

[0130] The eighth aspect relates to a food, a feed product, a pharmaceutical product, a dairy flavor or a cheese flavor product comprising the culture according to the second aspect or the third aspect.

[0131] The invention is further illustrated in the following non-limiting examples and figures.

[0132] Attached photos

[0133] Figure 1 is a graph showing dissolved oxygen (DO) curves of aerated bacteria grown in a medium without pH adjustment and with the addition of a heme supplement, according to an embodiment.

[0134] Figure 2 is a graph showing dissolved oxygen (DO) curves of aerating bacteria grown in a pH adjusted medium supplemented with a heme supplement according to an embodiment.

[0135] Figure 3 is a graph showing dissolved oxygen (DO) curves of aerated bacteria grown in a medium without pH adjustment and supplemented with a heme supplement according to an embodiment.

[0136] Figure 4 is a graph showing dissolved oxygen (DO) curves of aerating bacteria grown in a pH adjusted medium supplemented with a heme supplement according to an embodiment.

[0137] Figure 5 is a graph showing dissolved oxygen (DO) curves of aerated bacteria grown in a medium supplemented with a heme supplement, wherein the supplement has been sterile filtered ( Figure 5 A) or UHT treatment ( Figure 5 B).

[0138] Figure 6 is a graph showing dissolved oxygen (DO) curves of aerated bacteria grown in a medium supplemented with a heme supplement, wherein the supplement has been sterile filtered ( Figure 6 A) or UHT treatment ( Figure 6 B).

[0139] Figure 7is a graph showing dissolved oxygen (DO) curves of aerating bacteria grown in a medium supplemented with a heme supplement according to an embodiment.

[0140] Figure 8 is a graph showing the carbon dioxide evolution rate (CER) of aerating bacteria grown in a medium supplemented with a heme supplement, wherein the supplement has been sterile filtered ( Figure 8 A) or UHT treatment ( Figure 8 B).

[0141] Fig. 9 is a graph showing the oxygen uptake rate (OUR) of aerated bacteria grown in a medium supplemented with a heme supplement, wherein the supplement has been sterile filtered ( Fig. 9 A) or UHT treatment ( Fig. 9 B).

[0142] Fig.10 is a photograph of a sample taken during the measurement of the packed cell volume (PCV) % of an end of fermentation (EoF) broth sample of an aerated bacteria grown in a medium supplemented with a heme supplement, wherein the supplement has been sterile filtered ( Fig.10 A) or UHT treatment ( Fig.10 B).

[0143] Fig.11 is a measure of the viable cell count measured for cold broth samples at the end of fermentation (EoF) ( Fig.11 A) and acidification activity ( Fig.11 B) Bar graph showing changes in heme supplementation levels. Example

[0144] strains

[0145] The examples relate to the strains listed in Table 1. All strains have been deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the Leibniz-Institut DSMZ-German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, 38124 Braunschweig, Germany, with the accession numbers given in Table 1.

[0146] At the applicant's request, only samples of the deposited microorganisms described below may be provided to the expert, subject to the applicable regulations administered by the industrial property office of a Contracting State to the Budapest Treaty until the date of grant of the patent.

[0147] Table 1. Overview of strains used in the examples

[0148] strains Accession number Date of deposit Lactococcus lactis DSM 24648 2011-03-15

[0149] Medium preparation

[0150] In an embodiment, a bacterial growth medium containing non-protein-bound heme is used. It is referred to as non-protein-bound heme because heme is not bound to a protein containing a heme prosthetic group. This heme can be obtained by microbial expression, as described in EP3567109 (extracellular heme production method using metabolically engineered microorganisms).

[0151] Heme expressed according to the method described in EP3567109 is obtained as a supernatant powder or a biomass powder. The supernatant powder consists of a dried supernatant powder containing secreted heme at a concentration of about 1%-5% (w / w). The biomass powder consists of a dried cell material (again as a powder) containing non-secreted heme at a concentration of about 5%-10% (w / w). A stock solution is prepared by dissolving the required amount of heme-containing material from the dried supernatant or biomass powder in a 60mM NaOH aqueous solution to obtain a heme-rich stock solution with a final pH of typically 10-11. This is necessary because both powders are insoluble in water without increasing the pH level.

[0152] Example 1: Fermentation in BioLector

[0153] Stock solutions of both supernatant powder and biomass powder were prepared.Lactococcus lactis cells (DSM 24648) were grown in standard medium but with the addition of non-protein bound heme from either supernatant powder or biomass powder.A BioLector microbioreactor from Beckman Coulter was used according to standard instructions provided by the manufacturer.

[0154] Figure 1 is a graph showing dissolved oxygen (DO) profiles of aerated cell cultures in standard medium without pH adjustment (ie pH equal to 7.3) supplemented with sterile filtered heme from dried supernatant powder in a concentration range of 0.500% w / w - 0.010% w / w.

[0155] Figure 2 is a graph showing dissolved oxygen (DO) profiles of aerated cell cultures in standard medium adjusted to pH 6.3 and then supplemented with sterile filtered heme from dried supernatant powder at concentrations ranging from 0.033% w / w to 0.010% w / w.

[0156] This example shows that the heme contained in the dried supernatant powder is not able to support heme-induced respiration under aerobic conditions at any of the concentrations studied. Figure 1 and Figure 2No base consumption was observed under any of the conditions shown (error! Reference source not found). This was found to be associated with premature cessation of medium acidification, which may be associated with early growth arrest. Thus, a pH set point of 6.2 was never reached during any of the aerated cultures with heme from dried supernatant powder.

[0157] However, this example demonstrates that heme contained in dry biomass powder is able to support heme-induced respiratory growth in aerated culture. For high concentrations of dry biomass powder stock solutions, the onset of respiratory inhibition was observed.

[0158] In particular, two BioLector trials using filter-sterilized heme biomass powder showed that:

[0159] 1) When the pH of the cell culture medium is not adjusted (i.e., pH is about 7.3), respiratory metabolism occurs in the concentration range of 0.020%-0.008% w / w, such as Figure 3 shown.

[0160] 2) When the pH of the cell culture medium, which had previously been supplemented with sterile filtered heme (resulting in a starting pH in the range of 6.5-6.7), was adjusted to 6.3, respiratory metabolism occurred in the concentration range of 0.014%-0.010% w / w, e.g. Figure 4 shown.

[0161] For the heme concentration from the dry biomass powder when the medium pH is not adjusted, it is greater than 0.020% ( Figure 3 ), or for a medium previously supplemented with heme, the pH was adjusted to 6.3 with a concentration greater than 0.014% ( Figure 4), it was observed that iron-dependent heme cytotoxicity began to occur in the cells (Sawai, H., Yamanaka, M., Sugimoto, H., Shiro, Y., & Aono, S. (2012). Structural basis for the transcriptional regulation of hemehomeostasis in Lactococcus lactis. Journal of Biological Chemistry, 287(36), 30755–30768.; Joubert, L., Derré-Bobillot, A., Gaudu, P., Gruss, A., & Lechardeur, D. (2014). HrtBA and menaquinones control haem homeostasis in Lactococcus lactis. Molecular Microbiology, 93(4), 823–833.).

[0162] Hemin (olive green in aqueous solution; Tahoun, M., Gee, CT, McCoy, VE, Sander, PM, & Müller, CE (2021). Chemistry of porphyrins in fossil plants and animals. RSC Advances, 11(13), 7552–7563) consists of a ferric [Fe(III)] complex of protoporphyrin IX bound to chloride ions, which must be reduced to iron(II) to restore the ETC. The iron porphyrin should be a relatively stable complex, although prolonged interaction of hemin with oxygen may result in cleavage of the porphyrin ring, thereby removing the iron and its ability to react with cytochromes (Hogle, SL, Barbeau, KA, & Gledhill, M. (2014). Heme in the marine environment: From cells to the iron cycle. Metallomics, 6(6), 1107–1120). Protoporphyrin IX alone produces a reddish-brown aqueous solution (Tahoun et al., 2021). It can be speculated that oxidation of secreted heme contained in the dried supernatant powder may lead to the destruction of the heme molecule and thus also affect cell growth during aerobic cell culture in standard medium supplemented with sterile filtered heme from the dried supernatant powder.

[0163] Example 2: 2L bioreactor fermentation

[0164] Preparation of biomass powder stock solution Lactococcus lactis cells (DSM 24648) were grown in standard medium but with the addition of non-protein bound heme from biomass powder.

[0165] Fermentations were performed in 8 x 2 L fermenters in order to monitor online the evolution of the culture parameters of interest and to harvest the obtained microbial biomass to evaluate the performance of each culture in terms of PCV (%) and acidification activity.

[0166] Cultivation was carried out in a 2L Sartorius Biostat B bioreactor using cascade control, with agitator speed and air flow adjusted to maintain DO at a set point value of about 50%. The initial pH before inoculation was adjusted to 6.5, and then after the medium was acidified, the pH was maintained at a set point value of about 6.2 by automatic addition of alkali. The medium was supplemented with heme from dry biomass powder of 0.1% (w / w) sterile filtered (FM15, FM16, FM17 and FM18) or 0.1% (w / w) UHT treated (FM19, FM20, FM21 and FM22) stock solutions. The heme test concentration levels (i.e. 0.008%, 0.010%, 0.012% and 0.014%) of the two sterilized treatments were identical.

[0167] Fermentation conditions of the culture

[0168] Fermentation was performed in a 2L laboratory scale fermenter at 30°C with aeration using 1% (w / w) of the above culture as inoculum and one of the above non-protein bound heme as heme source. For aerobic fermentation as a positive control, the same conditions as aerobic fermentation were applied in a proprietary vegetarian friendly composite fermentation medium of Chr. Hansen A / S containing a non-vegetarian heme source and aeration. For anaerobic fermentation as a negative control, the same conditions as aerobic fermentation were applied in a proprietary vegetarian friendly composite fermentation medium of Chr. Hansen A / S without a heme source but without aeration.

[0169] The medium is sterilized by filtration or UHT treatment (141°C for 8-10 seconds). The finished medium has a pH of 6.5.

[0170] The culture was allowed to acidify to pH 6.0. The pH was subsequently maintained at 6.0 by controlled addition of 27% NH4OH.

[0171] When no further base consumption could be detected, the respective cultures were cooled to about 10°C.

[0172] After cooling, the bacteria in the culture medium were concentrated 6-18 times by centrifugation and then frozen into pellets in liquid nitrogen at one atmosphere to produce the so-called frozen direct-dive starter culture (F-DVS). The F-DVS pellets were stored at -50°C until further analysis.

[0173] Lactococcus lactis changes metabolism significantly when switching from anaerobic to respiratory growth. Biomass approximately doubles and acid production decreases during respiratory growth compared to anaerobic growth. A key feature of respiratory growth is the reduction of dissolved oxygen (DO, measured in %).

[0174] During the cultivation process, the following online parameters were monitored and recorded: DO ( Figure 5 ), alkali consumption rate ( Figure 6 ), total alkali consumption ( Figure 7 ), CER (Carbon Dioxide Release Rate) and OUR (Oxygen Uptake Rate) ( Figure 8 and Fig. 9 ). These measurements are used to evaluate whether and to what extent respiratory metabolism is established during the culture process.

[0175] Figure 5 Shown are dissolved oxygen (DO) curves for aerated cell cultures in standard medium supplemented with heme from sterile filtered (SF, upper panel, A) or UHT treated (lower panel, B) dry biomass powder. The heme test concentration levels (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) for both sterilization treatments were identical.

[0176] Figure 6 Base consumption rates of aerated cell cultures in standard medium supplemented with heme from sterile filtered (SF, upper panel, A) or UHT treated (lower panel, B) dry biomass powder are shown. The heme test concentration levels (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) for both sterilization treatments were the same.

[0177] Figure 7 Total base consumption of aerated cell cultures in standard medium supplemented with heme from sterile filtered (SF) or UHT treated dry biomass powder is shown. The heme concentration levels tested (i.e. 0.008%, 0.010%, 0.012% and 0.014%) were the same for both sterilization treatments.

[0178] Figure 8 Shown is the carbon dioxide evolution rate CER of aerated cell cultures in standard medium supplemented with heme from sterile filtered (SF, upper panel, A) or UHT treated (lower panel, B) dry biomass powder. The heme test concentration levels (i.e. 0.008%, 0.010%, 0.012% and 0.014%) for both sterilization treatments were the same.

[0179] Fig. 9 Shown is the oxygen uptake rate (OUR) of aerated cell cultures in standard medium supplemented with heme from sterile filtered (upper panel, A) or UHT treated (lower panel, B) dry biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) for both sterilization treatments were identical.

[0180] At the end of the cultivation, the respiratory performance of each aerobic culture was further evaluated by offline measurement of PCV (packed cell volume), viable cell count (by flow cytometry) and acidification activity (0.1% inoculum rate for all samples) on the final fermentation obtained, respectively. Fig.10 and Fig.11 shown.

[0181] Fig.10 Shown are packed cell volume (PCV) % measurements of end of fermentation (EoF) broth samples of aerated cell cultures with the addition of heme from sterile filtered (left, A) or UHT treated (right, B) dry biomass powder. The heme test concentration levels (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0182] Fig.11 Shown are active cell counts (upper panel, A) and acidification activity (lower panel, B) measured for end-of-fermentation (EoF) cold (i.e., <10°C) broth samples as a function of supplemented heme levels (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) for a first set of four aerobic cultures conducted using sterile filtered (SF) heme solutions and a second set of aerobic cultures conducted using UHT-treated heme solutions.

[0183] Under aerated conditions and in the presence of heme, respiratory metabolism occurred starting from 0.010% in the case of sterile-filtered heme-containing biomass powder, whereas aerobic respiration was observed in the entire concentration range tested for the UHT-treated powder. This may indicate that 0.008% filter-sterilized heme biomass powder is insufficient to fully activate bd-type cytochrome oxidase and therefore has a negative impact on the respiratory capacity of the cells.

[0184] Interestingly, it was observed that PCV (%) values ​​were greater with increasing concentration of sterile filtered heme biomass powder, whereas in case of UHT treated heme biomass powder, PCV values ​​were inversely correlated with the amount of heme supplement added to the culture medium ( Fig.10 ).

[0185] The acidification activity was consistent with the usual values ​​obtained by reference (typically Ta was about 88 min, data not shown), except for the case of cell culture in which the standard medium was supplemented with 0.008% filter-sterilized heme biomass powder (i.e. Ta equal to 113 min), as Fig.11 This is consistent with the fact that no effective breathing occurs in the last case.

[0186] According to this example, the best result in the 2L fermenter trial was a fermentate-PCV>9.5% and Ta=89min, which was observed for the heme biomass powder obtained from the UHT treated material at a concentration of 0.008% (w / w) powder. This concentration is equivalent to 80mg of heme powder per liter of culture medium, equivalent to 4ppm-8ppm of free heme. Translated to a larger scale, this would mean using 2.4kg of heme biomass powder for a full-scale 30m 3 Production of aeration batches.

[0187] This example demonstrates that sterile filtered and UHT treated stocks of dissolved biomass powders support respiration of a model Lactococcus strain.

[0188] For the material provided by sterile filtration, there was a dose response effect, showing that cell volume increased with increasing concentration of sterile filtered stock added. A similar trend was observed for viable cell number, as measured by flow cytometry (data not shown).

[0189] For the UHT treated material, a dose response effect was observed showing a decrease in cell volume with increasing concentration of UHT treated stock solution. A similar decreasing trend was observed for the number of viable cells as measured by flow cytometry (data not shown).

[0190] For the tested concentrations of dissolved dry biomass provided, all samples were able to achieve acceptable acidification performance when tested in a standardized dairy-based setting.

Claims

1. A method for obtaining a microbial culture, the method comprising the following steps: (i) culturing at least one microbial strain in a culture medium under aeration conditions and obtaining a fermentation product, (ii) harvesting the microbial strain from the fermentation product to obtain the microbial culture, wherein the culture medium comprises non-protein-bound heme.

2. The method of claim 1, wherein the non-protein-bound heme is produced by a microorganism.

3. The method of any one of claims 1 or 2, wherein the non-protein-bound heme is produced by expression of Aspergillus, Pichia, Bacillus, Saccharomyces or Escherichia.

4. A method according to any one of the preceding claims, wherein the non-protein bound heme is sterilized by filtration or heat treatment (such as UHT).

5. The method according to any one of the preceding claims, wherein the non-protein bound heme is added to achieve a concentration of about 0.1 g / kg fermentate to about 10 g / kg fermentate.

6. The method according to any one of the preceding claims, further comprising: (iii) concentrating the microbial culture to obtain a concentrated microbial culture.

7. The method according to any one of the preceding claims, further comprising: (iv) freezing or drying the microbial culture to obtain a frozen microbial culture or a dried microbial culture.

8. The method according to claim 6 or 7, further comprising: (v) packaging the frozen microbial culture or the dried microbial culture obtained in step (iv).

9. The method according to any one of the preceding claims, wherein the culture medium does not contain non-vegetarian compliant non-protein-bound heme.

10. The method according to any one of the preceding claims, wherein the microbial strain is Lactococcus lactis DSM 24648.

11. The method according to any one of the preceding claims, wherein the culture medium further comprises a thermostable compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers and non-ionic detergents.

12. A culture obtainable by the method according to any one of claims 1 to 11.

13. A culture or culture medium comprising at least one non-protein-bound heme.

14. A method for preparing a food, feed product, pharmaceutical, dairy flavor and cheese flavor product, the method comprising adding an effective amount of a culture according to claim 12 or 13 to a food, feed product or pharmaceutical starting material and maintaining the inoculated culture under conditions where the at least one microbial strain is metabolically active.

15. A fermented food, feed product or medicine, which can be obtained by the method according to any one of claims 1 to 11.

16. Use of at least one non-protein-bound heme in a fermentation method and / or a fermentation process.

17. A food, feed product, medicine, dairy flavor or cheese flavor product comprising the culture according to claim 12 or 13.

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