Method for preparing a culture of lactic acid bacteria, product, and medium therefor

Abstract

The present invention relates to a microbial starter culture. More specifically, a method for preparing a microbial culture, such as a lactic acid bacterium (LAB) starter culture, in which at least one microbial strain, such as a lactic acid bacterium, and at least one protein-unbound heme are inoculated into a 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. The microbial starter culture can be a lactic acid bacteria (LAB) starter culture in which lactic acid bacteria are inoculated into a medium containing at least one protein-unbound heme. This novel method utilizes raw materials that comply with the regulations for vegetarians. Therefore, the starter culture obtained by this new method is useful in the production of vegetarian foods, feeds, and pharmaceuticals.

Background Art

[0002] Microbial cultures are widely used not only in the food industry, feed industry, and pharmaceutical industry for producing fermented products including most dairy products such as cheese, yogurt, and butter, but also in meat products, bakery products, wine products, or vegetable products. Furthermore, microbial cultures are also used to produce proteins including enzymes and various types of useful compounds. Such microbial cultures are usually referred to as starter cultures, are produced in industrial growth plants, and are supplied to the fermentation brewing industry, for example, dairy farms, where the starter cultures are used in their production processes. In particular, cultures of lactic acid bacteria are widely used as starter cultures.

[0003] The production of lactic acid bacteria (LAB) starter cultures involves inoculating LAB cells into a specific fermentation medium in a cell number suitable for growing under appropriate fermentation conditions. In an industrial environment, as the end of the fermentation process approaches, a great deal of effort is being made to obtain the grown cells at a high concentration. The fermentation conditions and the fermentation medium need to support the growth of the cells in order to obtain the desired high biomass yield.

[0004] The methods currently used for the production of starter cultures of lactic acid bacteria, such as Lactococcus lactis starter cultures, utilize non-vegetarian compliant sources as raw materials in the fermentation medium. This non-vegetarian compliant source is utilized as an exogenous source. The exogenous source may be a heme source and is added to support the respiratory process of lactic acid bacteria. Since non-vegetarian compliant heme sources are being used, such starter cultures obtained by known methods cannot be used in vegetarian foods, feeds, and pharmaceuticals. Therefore, there is a need in the art to develop a respiratory process that utilizes a vegetarian compliant heme source for producing microbial starter cultures, such as lactic acid bacteria, with yields similar to those of processes known in the art.

[0005] As disclosed in the International Publication No. 2021 / 116311 (A1) pamphlet, yeast cells are introduced as a vegetarian compliant heme source. However, yeast cells are laborious to produce, process, and purify, which may lead to an increase in cost. In the case of yeast cells, dry matter other than LAB biomass is added. Using a yeast cell-based material in the form of yeast extract would add an extra processing step to the preparation of the protein-unbound heme-containing material.

[0006] The expression of heme using microorganisms can be carried out as shown, for example, in European Patent No. 3567109 entitled extracellular heme production method using metabolically engineered microorganism. However, it is unclear whether such heme can sustain cell cultures. SUMMARY OF THE INVENTION

[0007] The problem to be solved by the present invention is to provide a microbial culture such as a lactic acid bacterium culture applicable to the production of vegetarian foods, feeds, and pharmaceuticals.

[0008] Accordingly, a first aspect of the present invention is a method for obtaining a microbial culture, comprising: (i) culturing at least one microbial strain in a medium with aeration to obtain a fermented product; (ii) recovering the at least one microbial strain from the fermented product to obtain a microbial culture, wherein the medium contains at least one protein-unbound heme. The present invention relates to a method.

[0009] In a second aspect, the present invention relates to a culture obtainable by the method of the present invention.

[0010] In a third aspect, the present invention relates to a culture containing at least one protein-unbound heme.

[0011] A fourth aspect of the present invention relates to a medium containing at least one protein-unbound heme.

[0012] A fifth aspect of the present invention relates to a method for preparing a food product, a feed product, a pharmaceutical product, a dairy flavor, and a cheese flavoring product, comprising adding an effective amount of the culture of the present invention to a starting material for a food, a feed, or a pharmaceutical, and maintaining the inoculated culture under conditions where at least one type of microbial strain has metabolic activity.

[0013] A sixth aspect of the present invention relates to a fermented food, a fermented feed, or a fermented pharmaceutical obtainable by the method of the present invention.

[0014] The seventh aspect of the present invention relates to the use of at least one protein-unbound heme in a fermentation method and / or fermentation process.

[0015] The eighth aspect of the present invention relates to a food, feed, pharmaceutical, dairy-based flavor, or cheese-flavored product comprising the culture according to the second or third aspect.

Mode for Carrying Out the Invention

[0016] 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) in which protein-unbound heme is used as a vegetarian-friendly alternative heme source instead of a non-vegetarian heme source. When protein-unbound heme is applied as an exogenous heme source, surprisingly, it has been shown to assist the respiration of microbial strains (such as lactic acid bacteria). The purified protein-unbound heme is a vegetarian-friendly raw material. In this method, a yield comparable to methods known in the art is obtained.

[0017] Prior to discussing the detailed embodiments of the present invention, further definitions of selected terms used herein are provided.

[0018] As used herein, the term "protein-unbound heme" means free heme that is not bound to a protein containing a heme-deficient molecular family.

[0019] As used herein, the term "fermentation" refers to the process of growing or culturing microbial cells under aerobic or anaerobic conditions.

[0020] The term "starter culture" refers to a preparation containing microbial cells intended to be inoculated into the medium to be fermented.

[0021] As used herein, the term "yield" refers to the amount of biomass produced in the fermentation of a given volume. Yield can be measured in various ways; 1) as the biomass per unit volume measured by the absorbance (OD 600 ) at 600 nm of a 1 cm optical path length of the fermentation medium at the end of fermentation (background subtracted); 2) by the kg of the F-DVS culture at the end of fermentation, where the "acidification activity" or acidification power based on the Pearce test is 4.8 - 5.2; 3) by the packed cell volume (PCV) test, or; 4) by the cell count.

[0022] The term "F-DVS" refers to a so-called frozen direct vat set culture as described in the examples.

[0023] The legal framework regarding vegetarian claims in Europe is currently under revision and there are no unified rules at present. All claims based on European food law, vegan and vegetarian claims, are any messages or expressions that are not obligatory under the laws of the European Union or individual countries, and include any form of representation by pictures, graphs, or symbols that state, suggest, or imply that a food has certain characteristics (Neli Sochirca (2018), EFFL, 6, page 514). Thus, as used herein, the term "vegetarian-friendly heme source" refers to a heme source that is not obtained from or derived from animals and / or multicellular organisms. In contrast, the term "non-vegetarian heme source" refers to a heme source obtained from or derived from animals and / or multicellular structures.

[0024] In embodiments of the present invention, the one or more microbial strains are microbial strains that cannot perform respiratory growth without the addition of components / alternative components of the respiratory chain. It will be understood that the addition of components / alternative components of the respiratory chain may be the addition of an exogenous heme source.

[0025] At least one microbial strain is Lactococcus, Streptococcus, Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus, as currently known, Lactobacillus, Leuconostoc, Oenococcus, Weissella, Pediococcus,It may be selected from the group consisting of the genus Enterococcus, the genus Bifidobacterium, the genus Brevibacterium, the genus Propionibacterium, and combinations thereof. Although the majority of the genera in this group are "lactic acid bacteria", the industrially important genus is the genus Bifidobacterium, which may be included in the group of lactic acid bacteria because, although phylogenetically unrelated, lactic acid is one of the main fermentation end products. The list also includes other industrially important starter cultures belonging to the genera Brevibacterium and Propionibacterium, which are not included in the genera of lactic acid bacteria.,

[0026] As used herein, the term "lactic acid bacteria" (LAB) refers to Gram-positive, microaerophilic, or anaerobic bacteria that ferment sugars to produce acids including lactic acid (as the predominantly produced acid) and acetic acid. The industrially most useful lactic acid bacteria are those of the genera Lactococcus, Streptococcus, Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107.and Lactobacillus, Leuconostoc, Oenococcus, Weissella, Pediococcus, and Enterococcus, now known as Lentilactobacillus. Similarly, another industrially important genus is Bifidobacterium, which is phylogenetically unrelated but may be included in the group of lactic acid bacteria because lactic acid is one of the main fermentation end products.,

[0027] Thus, in one embodiment, at least one microbial strain is of the genus Lactococcus, Streptococcus, Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.Lactic acid bacteria selected from the group consisting of Lactobacillus, Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, Bifidobacterium, and combinations thereof, which are currently known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus as described in 004107.

[0028] The lactic acid bacteria strains that are generally used as LAB starter cultures are usually divided into mesophilic organisms with an optimal growth temperature of about 30°C and thermophilic organisms with an optimal growth temperature in the range of about 40 to about 45°C.

[0029] It will be understood that this Lactobacillus classification method was updated in 2020. This new classification method is disclosed in Zheng et al. 2020, and the classification methods important for the present invention are summarized below. [Table 1]

[0030] Typical organisms belonging to the mesophilic group include Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei (Lacticaseibacillus casei), and Lactobacillus paracasei subsp. paracasei (Lacticaseibacillus paracasei subsp. paracasei and Lacticaseibacillus paracasei subsp. tolerans). Examples of thermophilic lactic acid bacteria species include Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus.

[0031] Due to the fact that the content and thus the concentration of protein-unbound heme, lactic acid bacteria, protein-unbound heme, or any other nutrient in the medium can change over time, for example by uptake into microbial cells, it is necessary to refer to a specific point in time at which the concentration of protein-unbound heme must be measured or determined. Thus, the terms "initially" or "before fermentation" (which are used interchangeably herein) when used in relation to the concentration of protein-unbound heme, lactic acid bacteria, protein-unbound heme, or any other nutrient refer to the concentration of protein-unbound heme, lactic acid bacteria, protein-unbound heme, or any other nutrient present in the medium immediately before the microbial cells to be cultured are added to the medium.

[0032] However, throughout the fermentation process, it is also possible to add protein-unbound heme at any timing before harvest. The addition of protein-unbound heme can be carried out either batchwise or continuously. Thus, the "total addition amount" throughout the fermentation process becomes an important measure.

[0033] An important use of the starter culture according to the present invention is its use as so-called probiotics. In the present context, the term "probiotics" is understood as a microbial culture that, when ingested in live form by humans or animals, for example, suppresses harmful microorganisms in the gastrointestinal tract, enhances the immune system, or contributes to the digestion of nutrients, thereby bringing about an improvement in the state of health. A typical example of a product showing such probiotic activity is "sweet acidophilus milk".

[0034] The listing or discussion of a document that has clearly been previously published in this specification should not necessarily be construed as an admission that the document is part of the state of the art or common general knowledge.

[0035] Embodiments, preferences, and options regarding a given aspect, feature, or parameter of the present invention are to be considered disclosed in combination with all embodiments, preferences, and options regarding all other aspects, embodiments, features, and parameters of the present invention, unless the context indicates otherwise. For example, embodiments related to lactic acid bacteria cultures obtainable by the method of the present invention may equally apply to lactic acid bacteria starter cultures. Also, embodiments described in relation to the method of the present invention may relate to the products of the present invention, and vice versa.

[0036] Embodiments of the present invention are described below for illustrative purposes only.

[0037] A first aspect of the present invention is a method for obtaining a microbial culture, comprising: (i) culturing at least one microbial strain in a medium with aeration to obtain a fermented product; (ii) recovering the microbial strain from the fermented product to obtain a microbial culture. The medium contains at least one protein-unbound heme. The method relates to a method.

[0038] In an embodiment, the present invention is a method for obtaining a lactic acid bacteria culture, comprising: (i) culturing at least one lactic acid bacteria culture in a medium with aeration to obtain a fermented product; (ii) recovering the microbial strain from the fermented product to obtain a microbial culture. The medium contains at least one protein-unbound heme. The method relates to a method.

[0039] In one embodiment, the method of the present invention may further comprise the following steps: (iii) concentrating the microbial culture to obtain a concentrated microbial culture.

[0040] ​​ In one embodiment, the method of the present invention may further include the following steps: (iii) obtaining concentrated lactic acid bacteria by concentrating the lactic acid bacteria culture.

[0041] Concentration can be carried out using methods known in the art. These methods include, but are not limited to, centrifugation or ultrafiltration. In order to increase the number of microorganisms (such as lactic acid bacteria) in the concentrate obtained in step (iii), the concentration factor in step (iv) can be in the range of 2 to 20 (such as in the range of 6 to 19), for example in the range of 7 to 18 (such as in the range of 8 to 17), for example in the range of 9 to 16 (such as in the range of 10 to 15), for example in the range of 11 to 14 (such as in the range of 12 to 13), for example in the range of 2 to 4 (such as in the range of 3 to 6).

[0042] Commercially available starter cultures can generally be distributed as frozen cultures. At the low temperatures typically maintained by such frozen cultures, most metabolic activities in the cells cease, so it is possible to maintain the cells in this viable but dormant state for a long time.

[0043] Concentrated frozen cultures are of great commercial interest because they can be directly inoculated into production vessels. By using such concentrated frozen cultures, end-users can avoid the intermediate fermentation step, which would otherwise be inevitable and time-consuming, during which the starter culture would amplify. Additionally, end-users can significantly reduce the risk of contamination. Such concentrated cultures may be referred to as direct vat set (DVS) (trademark) cultures.

[0044] As an alternative to concentrated frozen cultures, concentrated freeze-dried direct vat set (DVS) (trademark) cultures (FD-DVS (trademark)) can be prepared. Such cultures have the additional advantage of being transportable without refrigeration.

[0045] Therefore, in an embodiment, the method of the present invention may further include the following steps: (iv) Obtaining a frozen microbial culture by freezing the microbial bacterial culture of step (ii) or the concentrated microbial culture of step (iii).

[0046] Thus, in an embodiment, the method of the present invention may further include the following steps: (iv) Obtaining a frozen lactic acid bacteria culture by freezing the lactic acid bacteria culture of step (ii) or the concentrated lactic acid bacteria culture of step (iii).

[0047] To remove liquid from the frozen microbial bacterial culture, the method of the present invention may further include the following steps: (v) Subliming water from the frozen microbial culture to obtain a dried microbial culture.

[0048] To remove liquid from the frozen lactic acid bacteria culture, the method of the present invention may further include the following steps: (v) Subliming water from the frozen lactic acid bacteria culture to obtain a dried lactic acid bacteria culture.

[0049] Step (v) can be carried out by a method selected from the group consisting of spray drying, spray freezing, vacuum drying, air drying, freeze drying, tray drying, and vacuum tray drying. In another embodiment, the method of the present invention further includes the following steps: (vii) Packaging the frozen microbial culture obtained in step (iv) or the freeze-dried microbial culture obtained in step (v).

[0050] It can be understood that the method of the present invention further includes the following steps: (vii) Packaging the frozen lactic acid bacteria culture obtained in step (iv) or the dried lactic acid bacteria culture obtained in step (v).

[0051] Damaging effects on the viability of living cells due to freezing and thawing are often observed. Generally, this damaging effect is considered to be due to cell dehydration and the formation of ice crystals in the cytosol during freezing.

[0052] However, a number of cryoprotective agents have been found that, for example, prevent or minimize ice crystallization in the cytosol upon freezing, such that freezing occurs in a controlled, minimally damaging manner.

[0053] Preferably, at least one cryoprotective substance is added to the recovered microbial culture obtained in step (ii), or to the recovered lactic acid bacteria culture, or to the concentrated microbial culture or concentrated lactic acid bacteria culture obtained in step (iii).

[0054] 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 compound. Examples of preferred cryoprotectants suitable for addition to the recovered microorganism essentially correspond to the preferred protein-unbound heme as described herein. The addition of such a cryoprotectant to the recovered microorganism is described in the patent application of the previously filed international application PCT / DK2004 / 000477. The preferred cryoprotectant described in PCT / DK2004 / 000477 is also the preferred cryoprotectant of the present invention. The complete description of PCT / DK2004 / 000477 is incorporated herein by reference. In a further preferred embodiment of the present invention, the 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-based or proteinaceous type cryoprotectants are generally not described as increasing the metabolic activity of thawed or reconstituted cultures. The cryoprotectant of the present invention can, in addition to cryoprotective activity, also result in an increase (booster effect) in the metabolic activity of the culture when inoculated into a fermentation, process treatment, or medium to be converted. Thus, one embodiment of the present invention is a frozen or dried culture in which the cryoprotectant is an agent or mixture of agents having a booster effect in addition to cryoprotective activity. The expression "booster effect" is used to describe a situation where a cryoprotectant results in an increase (booster effect) in the metabolic activity relative to a thawed or reconstituted culture when inoculated into a fermentation or converted medium. Viability and metabolic activity are not synonymous concepts. Commercially available frozen or dried (e.g., freeze-dried) cultures may retain viability but may have lost a significant portion of their metabolic activity. For example, the culture may lose its acid production (acidification) activity even over a short storage period. That is, viability and the booster effect need to be evaluated in different assays. Viability is evaluated by viability assays such as measurement of colony-forming units, while the booster effect is evaluated by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the viability of the culture.

[0055] The following acidification activity assay is an example of an assay that quantifies the associated metabolic activity of a thawed or re-prepared culture.

[0056] Although acid production activity is exemplified herein, the present invention is intended to encompass the stabilization of any type of metabolic activity of a culture. That is, the term "metabolic activity" refers to the oxygen removal activity of a culture, the acid production activity of a culture (i.e., production of, for example, lactic acid, acetic acid, formic acid, and / or propionic acid), or the production of metabolic products of a culture such as production of aromatic compounds such as acetaldehyde, (a-acetolactic acid, acetoin, diacetyl, and 2,3-butylene glycol (butanediol)).

[0057] In an embodiment of the present invention, the frozen culture contains, or comprises, a mixture of cryoprotectants or agents in an amount of 0.2% to 20% as % w / w of the frozen material ( contain ), or comprises ( comprise ). However, it is preferred to add a mixture of cryoprotectants or agents in an amount in the range of 0.2 wt% to 15 wt%, more preferably in the range of 0.2 wt% to 10 wt%, more preferably in the range of 0.5 wt% to 7 wt%, more preferably in the range of 1 wt% to 6 wt%, as measured as % w / w of the frozen material on a weight basis, for example, a mixture of cryoprotectants or agents in the range of 2 wt% to 5 wt%. In a preferred embodiment, the culture comprises an amount of a mixture of cryoprotectants or agents of approximately 3% as measured as % w / w of the frozen material on a weight basis. The preferred amount of approximately 3% cryoprotectant corresponds to a concentration in the range of 100 mM. It should be recognized that for each aspect of the embodiments of the present invention, the ranges can be increments of the recited ranges.

[0058] When the culture is a dry culture (e.g., lyophilized), it is preferred to add a cryoprotectant or a mixture of agents in an amount within the range of 0.8 wt% to 60 wt%, or within the range of 0.8 wt% to 55 wt%, or within the range of 1.3 wt% to 40 wt%, or within the range of 3 wt% to 30 wt%, or within the range of 6 wt% to 25 wt% (including the range of 10 wt% to 24 wt%) of the dry culture. In a preferred embodiment, the dry culture (e.g., lyophilized) contains approximately 16% of a cryoprotectant or a mixture of agents, measured as % w / w of the dry culture.

[0059] Furthermore, the frozen culture or the dry culture may contain additional conventional additives, such as nutrients including yeast extract, sugar, antioxidant, inert gas, and vitamins. Also, a surfactant containing a Tween® compound can be used as an additional additive to the culture according to the present invention. Further examples of such conventional additives that can be additionally added to the culture according to the present invention can be selected from proteins, protein hydrolysates, and amino acids. Preferred examples thereof 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.

[0060] More preferably, the conventional additive is a carbohydrate. Suitable examples thereof include pentoses (e.g., ribose, xylose), hexoses (e.g., fructose, mannose, sorbose), disaccharides (e.g., sucrose, trehalose, melibiose, lactulose), oligosaccharides (e.g., raffinose), fructooligosaccharides (e.g., Actilight, Fribrolose), polysaccharides (e.g., maltodextrin, xanthan gum, pectin, alginic acid, microcrystalline cellulose, dextran, PEG), and sugar alcohols (sorbitol, mannitol, and inositol) selected from the group consisting of.

[0061] Currently, the ratio (wt% / wt%) of at least one cryoprotectant to the concentrated microbial culture or the concentrated lactic acid bacteria culture is preferably in the range of 1:0.5 to 1:5, for example, in the range of 1:1 to 1:4 or 1:1.5 to 1:3, etc.

[0062] Another embodiment of the present invention is a method for preparing a microbial culture by increasing the yield as described herein, further comprising 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 method suitable for drying bacterial cultures.

[0063] At least one protein-unbound heme may be present in the medium or added to the medium before at least one microbial strain and / or lactic acid bacteria are added to the medium, or alternatively, at least one protein-unbound heme may be added immediately after at least one microbial strain and / or lactic acid bacteria are added to the medium.

[0064] In one embodiment, at least one protein-unbound heme is a protein expressed by a microorganism. In a preferred embodiment, the protein-unbound heme is the heme expressed according to the specification of European Patent No. 3567109 named extracellular heme production method using metabolically engineered microorganism.

[0065] In one embodiment, a composition comprising at least one protein-unbound heme is inactivated. To achieve the purpose of inactivating the native biological catalytic activity, several inactivation methods can be used, such as inactivation by pH (base), enzymatic digestion, or heat inactivation. In a preferred embodiment, the inactivation is heat inactivation. Heat inactivation may be carried out by any method known in the art, for example, autoclaving and / or UHT, etc., but is not limited thereto.

[0066] The inventors have surprisingly discovered that by adding a heat-stabilizing compound, it becomes possible to industrially process protein-unbound heme, and at the same time, a high growth yield becomes possible. In one embodiment, the medium further comprises a heat-stabilizing compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers, and nonionic surfactants. The heat-stabilizing compound may be selected from the group consisting of: sorbitol, glycerol, propylene glycol, mannitol, xylitol, propanediol, trehalose, sucrose, lactose, maltose, glucose, levan (fructose homopolysaccharide), dextran, dextran sulfate, gelatin (type A and type B), hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, fucoidan, poly sulfate pentosan, keratan sulfate, polyaspartic acid, polyglutamic acid, hydroxyethyl cellulose, 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, Bridgman 35 (polyoxyethylene alkyl ether). In a preferred embodiment, the heat-stabilizing compound is sorbitol.

[0067] In one embodiment, the protein-unbound heme is a protein that does not have native biological activity.

[0068] In one embodiment, the protein-unbound heme is produced by a microorganism. In one embodiment, the protein-unbound heme is indirectly derived from or directly produced by an organism of the genus Aspergillus, such as Aspergillus niger. In one embodiment, the protein-unbound heme is indirectly derived from or directly produced by an organism of the genus Pichia, such as Pichia pastoris. In one embodiment, the protein-unbound heme is indirectly derived from or directly produced by an organism of the genus Saccharomyces, such as Saccharomyces cerevisiae. In one embodiment, the protein-unbound heme is indirectly derived from or directly produced by an organism of the genus Escherichia, such as Escherichia coli. In one embodiment, the protein-unbound heme is indirectly derived from or directly produced by an organism of the genus Bacillus, such as non-spore-forming Bacillus.

[0069] At least one protein-unbound heme is added to the medium or present in the medium as a raw material intended to accelerate fermentation. The inventors have surprisingly discovered that the application of a non-vegetarian source in the medium can be replaced by at least one protein-unbound heme without reducing the yield.

[0070] Aspects of the invention thus relate to the use of at least one protein-unbound heme in a fermentation method and / or fermentation process.

[0071] The medium can be a complex fermentation medium.

[0072] The complex fermentation medium may be any complex fermentation medium known in the art, but the complex fermentation medium may contain compounds selected from the group consisting of lactose, nutrients, vitamins, tryptone, soya peptone, yeast extract, ascorbic acid, magnesium sulfate, milk, and combinations thereof.

[0073] In one embodiment, the protein-unbound heme is added at a level that allows respiration above the natural oxygen consumption level at which the cells can be maintained. The protein-unbound heme dose-dependently stimulates aerobic microbial growth such that oxygen consumption as a measure of microbial growth reaches a peak earlier and at a faster rate compared to culturing without said protein-unbound heme.

[0074] Thus, in one embodiment, oxygen consumption in the fermentate reaches its maximum value in less than 12 hours, such as less than 10 hours or less than 8 hours.

[0075] In one embodiment, oxygen consumption in the fermentate reaches 0.04 mol O2 / L / h in less than 10 hours or less than 8 hours.

[0076] Oxygen consumption can be measured using any method known to those skilled in the art.

[0077] In one embodiment, the medium in step (i) contains at least 0.005 w / w% of at least one protein-unbound heme, such as 0.008 w / w%, 0.01 w / w%, 0.014 w / w%, 0.03 w / w%, or 0.5 w / w%, etc., for example, within the range of 0.008 w / w% to 0.014 w / w%, within the range of 0.01 w / w% to 0.03 w / w%, within the range of 0.01 w / w% to 0.5 w / w%, or within the range of 0.005 w / w% to 0.5 w / w%, etc., of at least one protein-unbound heme, based on the weight of the medium (i.e., before at least one microbial strain is added) before fermentation (i.e., before at least one microbial strain is added).

[0078] In a preferred embodiment, the concentration of protein-unbound heme in the medium in step (i) is 0.008 w / w% to 0.014 w / w%.

[0079] A concentration of protein-unbound heme in the medium of about 0.008 w / w% (such as 0.008 w / w%) corresponds to about 9 to 10 ppm of heme and is particularly suitable for UHT sterilization.

[0080] A concentration of protein-unbound heme in the medium of about 0.014 w / w% (such as 0.014 w / w%) corresponds to about 16 to 17 ppm of heme and is particularly suitable for filter sterilization.

[0081] In another embodiment, the medium in step (i) contains, before fermentation, at least 0.5 w / w% of a microbial inoculum culture, for example, a lactic acid bacteria inoculum culture, based on the weight of the medium (i.e., before at least one microbial strain is added), for example, at least 1 w / w% (such as 1.5 w / w%) before fermentation, for example, 2 w / w% (such as 2.5 w / w%), for example, 3 w / w% (such as 3.5 w / w%), for example, 4 w / w%, for example, in the range of 0.5 to 4 w / w% (such as 1 to 3.5 w / w%), for example, 1.5 to 3 w / w% (such as 2 to 2.5 w / w%) of a lactic acid bacteria inoculum culture. The inoculum culture can be prepared according to the method described in Example 1.

[0082] In one embodiment, the protein-unbound heme is added at a concentration of about 0.1 g / kg of the fermented product to about 10 g / kg of the fermented product.

[0083] Surprisingly, in the method of the present invention, it may be possible to obtain a microbial culture such as a lactic acid bacteria culture that is sufficiently concentrated to be used for the production of F-DVS without concentrating the culture. However, even when this method is applied, most cultures need to be concentrated in order to obtain a starter culture that is commercially beneficial. Such cultures may preferably be recovered and concentrated by centrifugation or ultrafiltration.

[0084] Furthermore, in a preferred embodiment, the culture is carried out in a large fermenter containing 5 L to 100,000 L of a medium, preferably 300 L to 20,000 L of a medium.

[0085] A preferred embodiment is one in which the culture involves control of temperature and / or pH.

[0086] In an embodiment, the medium in step (i) and / or step (ii) contains one or more microbial strains that are microbial strains incapable of respiratory growth without supplementation with components / substitute components of the respiratory chain.

[0087] In an embodiment, the medium in step (i) and / or step (ii) is Lactococcus, Streptococcus, Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus, as currently known, Lactobacillus, Leuconostoc, Oenococcus,It contains at least one microbial strain selected from the group consisting of Weissella, Pediococcus, Enterococcus, Bifidobacterium, Brevibacterium, Propionibacterium, and combinations thereof.

[0088] In an embodiment, the medium in step (i) and / or step (ii) is Lactococcus, Streptococcus, Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.At least one lactic acid bacterium selected from the group consisting of Lactobacillus, Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, and Bifidobacterium, which are currently known as Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus as described in 004107 is included.

[0089] In an embodiment, the medium in step (i) and / or step (ii) contains one or more mesophilic organisms selected from the group consisting of Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei (new name: Lacticaseibacillus casei), Lactobacillus paracasei subsp. Paracasei ((Lacticaseibacillus paracasei subsp. paracasei and Lacticaseibacillus paracasei subsp. tolerans)), and Oenococcus oeni.

[0090] In another embodiment, the medium in step (i) and / or step (ii) contains one or more thermophilic organisms having an optimal growth temperature of about 40°C to about 45°C.

[0091] In an embodiment, the medium in step (i) and / or step (ii) contains one or more thermophilic organisms selected from the group consisting of Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus.

[0092] In an embodiment, the medium in step (i) and / or step (ii) is an LD culture containing one or more organisms selected from the group consisting of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis, and Leuconostoc mesenteroides subsp. cremoris. The term "LD culture" in the present context should be understood as a combination of Lactococcus lactis species and Leuconostoc species.

[0093] It should be understood that the medium in step (i) and / or step (ii) is an O-culture containing one or more organisms selected from the group including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. In the present context, "O-culture" should be understood as a medium containing Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. O-cultures are typically used to make hole-free cheeses (Cheddar, Cheshire, Feta). A specific culture is commercially available under the name R604 from Chr. Hansen A / S (Denmark) (Catalog No. 200113).

[0094] In a preferred embodiment, the medium in step (i) and / or step (ii) is a culture containing Lactococcus lactis.

[0095] To obtain the maximum yield, the recovery in step (ii) may preferably be carried out 5 to 24 hours after the start of the culture.

[0096] The method of the present invention may further include the preservation of the recovered 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).

[0097] Due to the high yield of this method, the microbial culture in the ferment obtained in step (i) is 2.0×10 10 ~5.0×10 10 active microbial cells per gram of the microbial culture, for example, 2.5×10 10 ~4.5×10 10 per gram of the microbial culture, for example, 3.0×10 10 ~4.0×1010 may contain viable microbial cells within a certain range. Similarly, the microbial culture in the fermented product obtained in step (i) contains 2.0×10 10 to 5.0×10 10 total microbial cells within a certain range per gram of the microbial culture, for example, 2.5×10 10 to 4.5×10 10 cells, for example, 3.0×10 10 to 4.0×10 10 total microbial cells within a certain range. From Table 2 in the experimental part, it can be seen that the number of viable 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 culture obtained by the present invention have high viability.

[0098] Due to the high yield of this method, the lactic acid bacteria culture in the fermented product obtained in step (i) contains 2.0×10 10 to 5.0×10 11 viable lactic acid bacteria cells within a certain range per gram of the lactic acid bacteria culture, for example, 2.5×10 10 to 4.5×10 10 cells, for example, 3.0×10 10 to 4.0×10 10 viable lactic acid bacteria cells within a certain range. Similarly, the lactic acid bacteria culture in the fermented product obtained in step (i) contains 2,0×10 10 to 5,0×10 10 total lactic acid bacteria cells within a certain range per gram of the acid bacteria culture, for example, 2.5×10 10 to 4.5×10 10 cells, for example, 3.0×10 10 to 4.0×10 10 total lactic acid bacteria cells within a certain range. From Table 2 in the experimental part, it can be seen that the number of viable 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 culture obtained by the present invention have high viability.

[0099] The viable cell count and / or total cell count is determined using flow cytometry, a technique known to those skilled in the art.

[0100] In a preferred embodiment, the increase in the yield of the microbial strain to be recovered, such as a lactic acid bacterium, or the microbial culture, such as a lactic acid bacterium culture, by this method is at least 1.2-fold, preferably at least 1.3-fold, more preferably at least 1.4-fold, even more preferably at least 1.5-fold, and most preferably at least 1.6-fold, compared to an anaerobic process excluding the heme source process.

[0101] 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, can be provided as a culture concentrate, such as a starter culture concentrate.

[0102] In a third aspect, the present invention relates to a microbial culture, such as a starter culture, containing at least one protein-unbound heme.

[0103] A fourth aspect relates to a medium containing at least one protein-unbound heme.

[0104] A fifth aspect of the present invention includes adding an effective amount of the culture according to the second or third aspect to a starting material for food, feed, or pharmaceuticals, and maintaining the inoculated culture under conditions where at least one type of microbial strain has metabolic activity, and relates to a method for preparing food, feed, pharmaceuticals, dairy-based flavors, and cheese flavor products.

[0105] Preferably, the food of the fifth aspect of the present invention is selected from the group consisting of milk-based products, vegetable products, meat products, beverages, fruit juices, wines, bakery products, dairy-based flavors, and cheese flavor products.

[0106] Preferably, the milk-based product is selected from the group consisting of cheese, yogurt, butter, inoculated sweet milk, and liquid fermented milk products.

[0107] In a sixth aspect, the present invention relates to a fermented food, fermented feed, or fermented pharmaceutical that can be obtained by the method of the first aspect.

[0108] A seventh aspect of the present invention relates to the use of at least one protein-unbound heme in a fermentation method and / or fermentation process.

[0109] An eighth aspect relates to a food, feed, pharmaceutical, dairy-based flavor, or cheese-flavored product comprising the culture described in the second or third aspect.

[0110] The present invention will be further described by the following non-limiting examples and drawings.

Brief Description of the Drawings

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Example

[0112] Strain Each example includes the strains listed in Table 1. All strains have been deposited with a depository institution that has obtained the status of an international depository authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany). The accession numbers are shown in Table 1.

[0113] The applicant requests that samples of the deposited microorganisms described below be made available only to experts until the date of patent grant, in accordance with the applicable regulations administered by the industrial property offices of the Contracting States to the Budapest Treaty. [Table 2]

[0114] Medium preparation In each example, a bacterial growth medium containing protein-unbound heme is used. Protein-unbound heme is so called because the heme is not bound to a protein containing a heme-deficient molecular family. Such heme can be obtained through expression by microorganisms as described in European Patent No. 3567109, Extracellular heme production method using metabolically engineered microorganism.

[0115] The heme expressed according to the method described in European Patent No. 3567109 was obtained as supernatant powder or biomass powder. The supernatant powder consists of the dried supernatant powder containing secreted heme at a concentration of approximately 1-5% (w / w). The biomass powder consists of the dried cell material (also in powder form) containing non-secreted heme at a concentration of approximately 5-10% (w / w). The stock solution was prepared by dissolving the desired amount of heme-containing material from the dried supernatant powder or dried biomass powder in an aqueous solution of 60 mM NaOH to obtain a heme-rich stock solution with a final pH usually between 10 and 11. This was necessary because neither of the two powders became soluble in water without raising the pH value.

[0116] Example 1: Fermentation in BioLector Stock solutions of both the supernatant powder and the biomass powder were prepared. Lactococcus lactis cells (DSM24648) were grown in a standard medium containing protein-unbound heme derived from the added supernatant powder or biomass powder. The BioLector microbioreactor manufactured by Beckman Coulter was used according to the standard instructions provided by the manufacturer.

[0117] Figure 1 is a graph showing the dissolved oxygen (DO) profiles of cell cultures under aeration in a standard medium supplemented with filter-sterilized heme from the dried supernatant powder at concentrations ranging from 0.500 to 0.010 w / w% without pH adjustment (i.e., pH equal to 7.3).

[0118] Figure 2 is a graph showing the dissolved oxygen (DO) profiles of cell cultures under aeration in a standard medium supplemented with filter-sterilized heme from the dried supernatant powder at concentrations ranging from 0.033 to 0.010 w / w% after adjusting the pH to 6.3.

[0119] This example shows that the heme contained in the dried supernatant powder was unable to support heme-induced respiration under aerobic conditions at any of the tested concentrations. No base consumption was observed under any of the conditions shown in Figures 1 and 2. This was observed in relation to the early arrest of the acidification of the culture broth and is presumably related to the initial growth arrest. As a result, the pH setpoint of 6.2 was not reached in any of the aerobic culture runs using heme from the dried supernatant powder.

[0120] However, this example shows that the heme contained in the dried biomass powder was able to support heme-induced respiratory growth in the aerated culture. Respiratory inhibition was observed in the high-concentration dried biomass powder stock solution. In particular, two BioLector tests using filter-sterilized heme biomass powder showed that respiratory metabolism occurred: 1) In the case where the pH of the cell medium was not adjusted (i.e., the pH was approximately 7.3) as shown in Figure 3, in the concentration range of 0.020 - 0.008 w / w%. 2) In the case where the pH of the cell medium was adjusted to 6.3 and then filter-sterilized heme was added (as a result, the starting pH value was 6.5 - 6.7) as shown in Figure 4, in the concentration range of 0.014 - 0.010 w / w%.

[0121] The expression of iron-dependent heme cytotoxicity in cells (Sawai, H., Yamanaka, M., Sugimoto, H., Shiro, Y., & Aono, S. (2012). Structural basis for the transcriptional regulation of heme homeostasis in Lactococcus lactis. Journal of Biological Chemistry, 287(36), 30755 - 30768.; Joubert, L., Derre-Bobillot, A., Gaudu, P., Gruss, A., & Lechardeur, D. (2014). HrtBA and menaquinones con-trol haem homeostasis in Lactococcus lactis. Molecular Microbiology, 93(4), 823 - 833.) was observed when the concentration of heme from dry biomass powder was greater than 0.020% when the pH of the medium was not adjusted (Figure 3), or when the concentration was greater than 0.014% when the pH of the medium was adjusted to 6.3 and then heme was added (Figure 4).

[0122] Hemin (yellow-green in aqueous solution; Tahoun, M., Gee, C. T., McCoy, V. E., Sander, P. M., & Muller, C. E. (2021). Chemistry of porphyrins in fossil plants and animals. RSC Advances, 11(13), 7552-7563) consists of a trivalent iron [Fe(III)] complex of protoporphyrin IX bound to chloride ions and needs to be reduced to iron(II) in order to regenerate the ETC. Iron porphyrin should be a relatively stable complex, but if the interaction between hemin and oxygen persists, the porphyrin ring may be broken, thereby removing iron and potentially removing the ability to react with cytochrome (Hogle, S. L., Barbeau, K. A., & Gledhill, M. (2014). Heme in the marine environment: From cells to the iron cycle. Metallomics, 6(6), 1107-1120). Protoporphyrin IX alone gives a reddish-brown aqueous solution (Tahoun et al., 2021). It can be speculated that the oxidation of secreted heme contained in the dry supernatant powder also affected cell growth during aerobic culture of cells in a standard medium supplemented with filter-sterilized heme from the dry supernatant powder because the hemin molecules were destroyed.

[0123] Example 2: 2L Bioreactor Fermentation A stock solution of biomass powder was prepared. Lactococcus lactis cells (DSM24648) were grown in a standard medium containing protein-unbound heme derived from the added biomass powder.

[0124] To evaluate the performance of each culture with respect to PCV (%) and acidification activity, the progress of the culture parameters of interest was monitored online, and fermentation was carried out in eight 2L fermenters to recover the resulting microbial mass.

[0125] The cultivation was carried out using cascade control in which both the agitator speed and the air flow were controlled to maintain the DO at around 50% of the set value in a 2 L Sartorius Biostat B bioreactor. The starting pH before inoculation was adjusted to 6.5, and then after the medium acidified, the pH was maintained at around 6.2 of the set value during automatic base addition. Heme from dried biomass powder was added to the medium as 0.1% (w / w) of a filter-sterilized stock solution (FM15, FM16, FM17, and FM18) or 0.1% (w / w) of a UHT-treated stock solution (FM19, FM20, FM21, and FM22). The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0126] Fermentation conditions of the culture Fermentation was carried out at 30 °C under aeration in a 2 L laboratory-scale fermenter, using 1% (w / w) of the above culture as the inoculum and one of the above protein-unbound hemes as the heme source. For aerobic fermentation as a positive control, the same conditions as in the case of aerobic fermentation were applied, and it was carried out in a vegetarian-compatible complex fermentation medium patented by Chr. Hansen containing a non-vegetarian heme source under aeration. For anaerobic fermentation as a negative control, the same conditions as in the case of aerobic fermentation were applied, except that aeration was not carried out and it was carried out in a vegetarian-compatible complex fermentation medium patented by Chr. Hansen that does not contain a heme source.

[0127] The medium was sterilized by filtration or UHT treatment (141 °C for 8 - 10 seconds). The pH of the medium at the end was 6.5.

[0128] The culture was acidified to pH 6.0. The pH was subsequently maintained at 6.0 by controlled addition of 27% NH4OH.

[0129] When no further base consumption was detected, each culture was cooled to approximately 10 °C.

[0130] After cooling, the bacteria in the medium were concentrated 6 - 18 times by centrifugation and then frozen as pellets in liquid nitrogen at 1 atmosphere to prepare a so-called Frozen Direct Vat Set (F-DVS) culture. This F-DVS pellet was stored at -50 °C until further analysis.

[0131] When Lactococcus lactis transitions from anaerobic growth to respiratory growth, its metabolism changes significantly. Compared to anaerobic growth, during respiratory growth, the biomass approximately doubles and acid production decreases. An important feature of respiratory growth is the decrease in dissolved oxygen (DO, measured in % units).

[0132] During the culturing process, the following online parameters were monitored and recorded: DO (Figure 5), base consumption rate (Figure 6), total base consumption (Figure 7), CER (carbon dioxide evolution rate), and OUR (oxygen uptake rate) (Figures 8 and 9). These measurements were used to evaluate whether and to what extent respiratory metabolism was established during the culturing process.

[0133] Figure 5 shows the dissolved oxygen (DO) profiles for cell cultures under aeration in a standard medium supplemented with heme from filter-sterilized (SF, upper graph, A) or UHT-treated (lower graph, B) dried biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0134] Figure 6 shows the base consumption rate for cell cultures under aeration in a standard medium supplemented with heme from filter-sterilized (SF, upper graph, A) or UHT-treated (lower graph, B) dried biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0135] Figure 7 shows the total base consumption in cell culture under aeration in a standard medium supplemented with heme from filter-sterilized (SF) or UHT-treated dry biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0136] Figure 8 shows the carbon dioxide evolution rate CER in cell culture under aeration in a standard medium supplemented with heme from filter-sterilized (SF, upper graph, A) or UHT-treated (lower graph, B) dry biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0137] Figure 9 shows the oxygen uptake rate (OUR) in cell culture under aeration in a standard medium supplemented with heme from filter-sterilized (upper graph, A) or UHT-treated (lower graph, B) dry biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0138] Once the culture was completed, the respiratory performance of each aerobic culture was further evaluated for the resulting final fermentate by offline measurements of PCV (packed cell volume), viable cell count (via flow cytometry), and acidification activity, as shown in Figures 10 and 11, respectively (inoculation rate 0.1% for all samples).

[0139] Figure 10 shows the measured values of packed cell volume (PCV)% of culture broth samples at the end of fermentation (EoF) from cell culture under aeration performed with heme from filter-sterilized (left hand side, A) or UHT-treated (right hand side, B) dry biomass powder. The heme concentration levels tested (i.e., 0.008%, 0.010%, 0.012%, and 0.014%) were the same for both sterilization treatments.

[0140] Figure 11 shows, for both a first set consisting of four aerobic cultures carried out using a filter-sterilized (SF) heme solution and a second group consisting of aerobic cultures carried out using a UHT-treated heme solution, the viable cell count (upper figure, A) and acidification activity (lower figure, B) measured on cold (i.e., <10 °C) culture broth samples at the end of fermentation (EoF) as a function of the added heme level (i.e., 0.008%, 0.010%, 0.012%, and 0.014%).

[0141] Under aerated conditions and in the presence of heme, in the case of filter-sterilized heme-containing biomass powder, respiratory metabolism started from 0.010%, while in the case of UHT-treated powder, aerobic respiration was observed over the entire range of concentrations tested. This may indicate that 0.008% filter-sterilized heme biomass powder was insufficient to fully activate bd-type cytochrome oxidase, and as a result, it had a negative impact on the respiratory ability of the cells.

[0142] Interestingly, for filter-sterilized heme biomass powder, the higher the concentration, the greater the PCV (%) value obtained, while in the case of UHT-treated heme biomass powder, the PCV value was found to be inversely proportional to the amount of heme supplement added to the culture medium (Figure 10).

[0143] As shown in the lower figure of Figure 11, the acidification activity was consistent with the normal values obtained using a reference (typically, Ta was about 88 minutes, data not shown), except for the case of cell cultures with 0.008% filter-sterilized heme biomass powder added to the standard medium (i.e., Ta = 113 minutes). This was consistent with the fact that no effective respiration occurred in the case of 0.008% filter-sterilized heme biomass powder.

[0144] The best results in the 2L fermenter test according to this example were a fermentate - PCV > 9.5% and Ta = 89 minutes, which were observed in the case of the heme biomass powder obtained with a UHT - treated material at a concentration of 0.008% (w / w) powder. This concentration was equivalent to 80 mg of heme powder per liter of culture medium and corresponded to 4 - 8 ppm of free heme. Scaling up, this would mean using 2.4 kg of heme biomass powder for the production in a full - scale 30 m 3 aerated batch.

[0145] This example shows that both filter - sterilized and UHT - treated stock solutions of dissolved biomass powder supported the respiration of the Lactococcus strain model.

[0146] For the material obtained by filter sterilization, a dose - response effect was shown where the cell volume increased as the concentration of the added filter - sterilized stock solution increased. A similar trend was also observed for the number of viable cells measured by flow cytometry (data not shown).

[0147] For the UHT - treated material, a dose - response effect was shown where the cell volume decreased as the concentration of the UHT - treated stock solution increased. A similar decreasing trend was also observed for the number of viable cells measured by flow cytometry (data not shown).

[0148] At the test concentrations of the provided lyophilized biomass, when tested in a standardized milk - based setting, all samples were able to allow acceptable acidification performance.

Claims

1. A method for obtaining a microbial culture, (i) A step of culturing at least one microbial strain in a culture medium while aerating to obtain a fermented product, (ii) A step of obtaining the microbial culture by recovering the microbial strain from the fermented product, Includes, The culture medium contains protein-unbound heme. method.

2. The method according to claim 1, wherein the protein-unbound heme is produced by a microorganism.

3. The method according to any one of claims 1 or 2, wherein the protein-unbound heme is produced by expression from the genera Aspergillus, Pichia, Bacillus, Saccharomyces, or Escherichia.

4. The method according to either claim 1 or 2, wherein the protein-unbound heme is sterilized by filtration or heat treatment (such as UHT).

5. The method according to either claim 1 or 2, wherein the protein-unbound heme is added to a concentration of approximately 0.1 g / kg of ferment to approximately 10 g / kg of ferment.

6. (iii) Concentrate the microbial culture to obtain a concentrated microbial culture. The method according to any one of claims 1 or 2, further comprising:

7. (iv) Freezing or drying the microbial culture to obtain a frozen microbial culture or a dried microbial culture. The method according to any one of claims 1 or 2, further comprising:

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

9. The method according to any one of claims 1 or 2, wherein the culture medium does not contain protein-unbound heme that is not suitable for vegetarians.

10. The method according to either claim 1 or 2, wherein the microbial strain is Lactococcus lactis DSM 24648.

11. The method according to any one of claims 1 or 2, wherein the culture medium further comprises a heat-stabilized compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers, and nonionic surfactants.

12. A culture that can be obtained by the method described in either claim 1 or 2.

13. A culture or medium containing at least one protein-unbound heme.

14. A method for preparing food, feed, pharmaceuticals, dairy flavorings, and cheese flavoring products, comprising adding an effective amount of the culture described in claim 12 to a starting material for food, feed, or pharmaceuticals, and maintaining the inoculated culture under conditions in which at least one microbial strain has metabolic activity.

15. A method for preparing food, feed, pharmaceuticals, dairy flavorings, and cheese flavoring products, comprising adding an effective amount of the culture described in Claim 13 to a starting material for food, feed, or pharmaceuticals, and maintaining the inoculated culture under conditions in which at least one microbial strain has metabolic activity.

16. A fermented food, a fermented feed, or a fermented pharmaceutical that can be obtained by the method described in either claim 1 or 2.

17. The use of at least one protein-unbound heme in fermentation methods and / or fermentation processes.

18. A food, feed, pharmaceutical, dairy-based flavoring, or cheese-flavored product comprising the cultured product described in claim 12.

19. A food, feed, pharmaceutical, dairy-based flavoring, or cheese-flavored product comprising the cultured product described in Claim 13.

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