Method for producing culture of lactic acid bacteria, its product and culture medium

Abstract

The present invention relates to microbial starter cultures, and more particularly to a method for preparing microbial cultures, such as lactic acid bacteria (LAB) starter cultures, by inoculating a culture medium with at least one microbial strain, such as lactic acid bacteria, and at least one heme protein.

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 is a lactic acid bacteria (LAB) starter culture, where the lactic acid bacteria are inoculated into a culture medium, and the culture medium comprises at least one heme protein. The novel method uses raw materials that comply with vegetarian regulations. Thus, the starter culture obtained by the novel method is useful for the production of vegetarian food, feed, and medicines. [Background technology]

[0002] Microbial cultures are widely used in the food, feed and pharmaceutical industries in the production of fermented products such as most dairy products, such as cheese, yogurt and butter, as well as meat, bread, wine and vegetable products. In addition, microbial cultures are also used for the production of proteins, including enzymes and various useful compounds. Such microbial cultures are usually called starter cultures, which are produced in industrial propagation plants and distributed to fermentation industries, such as dairy plants, 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 with an appropriate number of cells to grow under the appropriate fermentation conditions. In industrial settings, significant efforts are made to achieve a high concentration of grown cells at the end of the fermentation process. To obtain the desired high biomass yield, the fermentation conditions and fermentation medium must support cell growth.

[0004] Currently, the methods used for producing lactic acid bacteria starter cultures, such as Lactococcus lactis starter cultures, use non-vegetarian raw materials as the source of the fermentation medium. The non-vegetarian raw materials are used as exogenous raw materials. The exogenous raw materials may be a source of heme and are added to support the respiration process of the lactic acid bacteria. Due to the use of a non-vegetarian heme source, such starter cultures obtained by known methods are not applicable for vegetarian foods, feeds and medicines. Thus, there is a need in the art to develop a respiration process for producing microbial starter cultures, for example of lactic acid bacteria, applying a vegetarian heme source with a yield similar to that of the processes known in the art.

[0005] As disclosed in International Publication No. WO2021 / 116311A1, yeast cells have been implemented as a vegetarian source of heme. However, yeast cells are laborious to produce, process and purify, which can increase costs. Yeast cells add dry matter in addition to the LAB biomass. The use of yeast cell-based material in the form of yeast extract adds an additional processing step to the preparation of heme protein-containing material. Summary of the Invention [Problem to be solved by the invention]

[0006] The problem to be solved by the present invention is to provide a microbial culture, such as a lactic acid bacteria culture, that can be applied to the production of plant-based foods, feeds, and pharmaceuticals. [Means for solving the problem]

[0007] Thus, according to a first aspect, the present invention provides a method for producing ... medicament for use in a pharmaceutical composition comprising the steps of: (i) culturing at least one microbial strain in a culture medium under aeration to obtain a fermentate, wherein said culture medium contains at least one heme protein; (ii) harvesting the microbial strain from the fermentation to obtain a microbial culture; The present invention relates to a method for obtaining a microbial culture, comprising:

[0008] According to a second aspect, the present invention relates to a culture obtainable by the method of the invention.

[0009] According to a third aspect, the present invention relates to a culture comprising at least one hemoprotein.

[0010] According to a fourth aspect, the present invention relates to a culture medium comprising at least one heme protein.

[0011] A fifth aspect of the invention relates to a method for producing food, feed, pharmaceutical, dairy flavour and cheese flavour products, the method comprising adding an effective amount of a culture of the invention to a food, feed or pharmaceutical starting material and maintaining the inoculated culture under conditions in which at least one microbial strain is metabolically active.

[0012] A sixth aspect of the present invention relates to a fermented food, feed or pharmaceutical product obtainable by the method of the present invention.

[0013] A seventh aspect of the invention relates to the use of at least one heme protein in a fermentation method and / or process.

[0014] An eighth aspect of the invention relates to a food, feed, pharmaceutical, dairy flavour or cheese flavour product comprising a culture according to the second or third aspect. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 illustrates oxygen transfer rates according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The inventors have developed a method for obtaining microbial cultures, such as starter cultures of microbial strains (e.g., Lactobacillus subtilis), using hemoproteins as a vegetarian alternative heme source instead of non-vegetarian heme sources. Application of hemoproteins as an exogenous heme source has surprisingly been shown to support respiration of microbial strains (such as Lactobacillus subtilis). Purified hemoproteins are vegetarian raw materials. This method results in yields comparable to methods known in the art.

[0017] Before describing detailed embodiments of the invention, further definitions of selected terms used herein are provided.

[0018] As used herein, the term "fermentation" refers to the process of growing or culturing microbial cells under aerobic or anaerobic conditions.

[0019] The term "starter culture" refers to a preparation containing microbial cells intended to inoculate a fermentation medium.

[0020] Here, the term "yield" refers to the amount of biomass produced in a given volume of fermentation. Yield can be measured in a variety of ways, but here we measure it in two different ways: 1) as biomass per unit volume (background subtracted) measured by optical density at 600 nm in a 1 cm light path (OD600) of the fermentation medium at the end of the fermentation; 2) by an "acidifying activity" or acidifying power of 4.8-5.2 by Pierce test, per kg of F-DVS culture at the end of the fermentation; 3) by the packed cell volume (PCV) test; or 4) by cell number.

[0021] The term "F-DVS" refers to so-called frozen direct vat set cultures, as explained in the Examples.

[0022] The European legal framework on vegetarian labelling is currently under revision and there are currently no uniform rules. All labelling under European food legislation, vegan labelling and vegetarian labelling are messages or representations not required by European Union or national law, including any form of pictorial, graphical or symbolic representation that states, suggests or implies that a food has certain properties (Neli Sochirca (2018), EFFL, 6, p. 514). Thus, in this context, the term "vegetarian 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 that is obtained from or derived from animals and / or multicellular structures.

[0023] According to one embodiment of the invention, the one or more microbial strains are microbial strains that are incapable of respiratory growth without supplementation of a component / alternative of the respiratory chain, it being understood that supplementation of a component / alternative of the respiratory chain may be supplementation of an exogenous source of heme.

[0024] The at least one microbial strain may be selected from the group consisting of: Lactococcus, Streptococcus, Lactobacillus, currently known as Ligilactobacillus, Holzapfelia, mylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacilus, Lactococcus spp. ... lus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus (Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc., Oenococcus., Leuconostoc.), Pediococcus, Enterococcus, Bifidobacterium, Brevibacterium, Propionibacterium and combinations thereof. Most of the genera in this group are "lactic acid bacteria", but the industrially important genus Bifidobacterium, although phylogenetically unrelated, is sometimes included in the group of lactic acid bacteria because 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 genus Lactobacillus.

[0025] As used herein, the term "lactic acid bacterium" (LAB) refers to gram-positive, microaerophilic or anaerobic bacteria that ferment sugars to produce acids, including lactic acid (the primary acid produced) and acetic acid. The most industrially useful lactic acid bacteria are found in the following genera: Lactococcus, Streptococcus, Lactobacillus now known as Lactobacillus, Holzapfelia, mylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, La. Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus (Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc., Oenococcus, Leuconostoc., Weissella, Pediococcus, and Enterococcus. As mentioned above, another genus of industrial importance is the Bifidobacteria, which, although phylogenetically unrelated, are sometimes included in the group of lactic acid bacteria, since lactic acid is one of the main fermentation end products.

[0026] Thus, according to one embodiment, the at least one microbial strain is a lactic acid bacterium selected from the group consisting of Lactococcus, Streptococcus, Lactobacillus, currently known as Ligilactobacillus, Holzapfelia, mylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacti caseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus (Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc., Oenococcus., Leuconostoc.), Pediococcus, Enterococcus, Bifidobacterium, and combinations thereof.

[0027] Commonly used LAB starter culture strains of lactic acid bacteria are generally divided into mesophilic bacteria, whose optimum growth temperature is about 30°C, and thermophilic bacteria, whose optimum growth temperature is in the range of about 40 to about 45°C.

[0028] It will be appreciated that the classification of the Lactobacillus genus was updated in 2020. The new classification is disclosed in Zheng et al. 2020 and those of importance to the present invention are summarized below.

[0029] [Table 1]

[0030] Representative 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. paracasei (Lacticaseibacillus paracasei subsp. paracasei and Lacticaseibacillus paracasei subsp. tolerans). Thermophilic lactic acid bacteria species include, for example, Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus.

[0031] Since the amount, and therefore the concentration, of heme protein, lactic acid bacteria, heme protein, or other nutrient in the medium may change over time, such as due to uptake into the microbial cells, it is necessary to refer to the specific point in time at which the concentration of the heme protein needs to be measured or determined. Thus, the terms "initially" or "pre-fermentation" (used interchangeably herein) used in relation to the concentration of heme protein, lactic acid bacteria, heme protein, or other nutrient in the medium refer to the concentration of the heme protein, lactic acid bacteria, heme protein, or other nutrient present in the medium immediately before the microbial cells to be cultured are added to the medium.

[0032] However, it is also possible to add hemoproteins at any time during the fermentation process before harvest. Hemoprotein addition can be done in a batch or continuous manner. Therefore, the "total amount added" throughout the fermentation process is one of the important indicators.

[0033] An important application of the starter cultures according to the invention are so-called probiotics. In the present specification, the term "probiotics" is understood as microbial cultures which, when ingested by humans or animals in the form of live cells, improve the health status, for example by suppressing harmful microorganisms in the gastrointestinal tract, by strengthening the immune system or by contributing to the digestion of nutrients. A typical example of such a probiotically active product is "sweet acidophilus milk".

[0034] The listing or discussion of an apparently prior-published document in this specification does not necessarily imply an acknowledgement that the document is part of the state of the art or is common knowledge.

[0035] Embodiments, preferences and options relating to a particular aspect, feature or parameter of the invention should be considered as disclosed in combination with all embodiments, preferences and options relating to all other aspects, embodiments, features and parameters of the invention, unless the context dictates otherwise. For example, an embodiment relating to a lactic acid bacteria culture obtained by a method of the invention may be applied to a lactic acid bacteria starter culture as well. Also, an embodiment described in relation to a method of the invention may be related to a product of the invention and vice versa.

[0036] Embodiments of the present invention are described below by way of example only.

[0037] One aspect of the present invention relates to a process for producing a method for producing a pharmaceutical composition comprising the steps of: (i) culturing at least one microbial strain in a culture medium under aeration to obtain a fermentate, wherein said culture medium contains at least one heme protein; (ii) harvesting the microbial strain from the fermentation to obtain a microbial culture; The present invention relates to a method for obtaining a microbial culture, comprising:

[0038] According to one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: (i) culturing at least one lactic acid bacteria culture in a culture medium under aeration to obtain a fermentate, wherein said culture medium contains at least one heme protein; (ii) harvesting the microbial strain from the fermentation to obtain a microbial culture; The present invention relates to a method for obtaining a lactic acid bacteria culture, comprising the steps of:

[0039] According to one embodiment, the method of the invention comprises the steps of: (iii) concentrating the microbial culture to obtain an enriched microbial culture.

[0040] According to one embodiment, the method of the invention comprises the steps of: (iii) concentrating the lactic acid bacteria culture to obtain a concentrated lactic acid bacteria culture.

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

[0042] Commercially available starter cultures are generally available as frozen cultures, and the low temperatures at which such cultures are typically maintained cause most of the metabolic activity within the cells to cease, and the cells to remain in this suspended (but viable) state for extended periods of 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, the end user can avoid the normally mandatory and time-consuming intermediate fermentation step while the starter culture is amplified, and furthermore, the risk of contamination is significantly reduced. Such concentrated cultures are sometimes called DVS (direct vat set® cultures).

[0044] As an alternative to concentrated frozen cultures, concentrated freeze-dried direct vat set cultures, FD-DVS, can also be prepared. Such cultures have the added advantage that they can be shipped without refrigeration.

[0045] Thus, according to one embodiment, the method of the invention may further comprise the steps of: (iv) freezing the microbial bacterial culture of step (ii) or the concentrated microbial culture of step (iii) to obtain a frozen microbial culture.

[0046] Thus, according to one embodiment, the method of the invention may further comprise the steps of: (iv) Freezing the lactic acid bacteria culture of step (ii) or the concentrated lactic acid bacteria culture of step (iii) to obtain a frozen lactic acid bacteria culture.

[0047] To remove liquid from a frozen microbial culture, the method of the invention may further comprise the steps of: (v) sublimating 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 invention may further comprise the steps of: (v) Sublimating water from the frozen lactic acid bacteria culture to obtain a dried lactic acid bacteria culture.

[0049] Step (v) may be carried out 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. According to a further embodiment, the method of the present invention further comprises the step of: (vii) packaging said frozen microbial culture obtained in step (iv) or the freeze-dried microbial culture obtained in step (v).

[0050] It will be appreciated that the method of the present invention further comprises the steps of: (vii) packaging the frozen lactic acid bacteria culture obtained in step (iv) or the dried lactic acid bacteria culture obtained in step (v).

[0051] Freezing and thawing has often been observed to adversely affect the viability of living cells, which is generally believed to be due to dehydration of the cells during freezing and the formation of ice crystals within the cytoplasm.

[0052] However, a number of cryoprotectants have been discovered that allow freezing to be controlled and damage minimized, including preventing or minimizing ice crystallization within the cytoplasm during freezing.

[0053] Preferably, at least one cryoprotectant is added to the harvested microbial culture or the harvested lactic acid bacteria culture obtained in step (ii) or to the concentrated microbial culture or the 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 the biosynthesis of nucleic acids, or one or more derivatives of such compounds. Examples of preferred cryoprotectants suitable for addition to the harvested microorganisms essentially correspond to the preferred heme proteins described herein. Addition of such cryoprotectants to the harvested microorganisms is described in the previously filed patent application with application number PCT / DK2004 / 000477. The preferred cryoprotectants described in PCT / DK2004 / 000477 are also preferred cryoprotectants of the present invention. The full description in PCT / DK2004 / 000477 is incorporated herein by reference. According to a further preferred embodiment of the present invention, the one or more cryoprotectants are selected from the group of nucleoside monophosphates. According to a preferred embodiment, the at least one or only cryoprotectant is an IMP. Carbohydrate or protein type cryoprotectants have not generally been described to increase the metabolic activity of thawed or reconstituted cultures. In addition to their cryoprotective activity, the cryoprotectants of the present invention can also increase the metabolic activity (booster effect) of the culture when the culture is inoculated into a medium to be fermented, processed or converted. Thus, one embodiment of the present invention is a frozen or dried culture, and the cryoprotectant is an agent or mixture of agents that has a booster effect in addition to cryoprotective ability. The expression "booster effect" is used to describe the situation where a cryoprotectant confers an increased metabolic activity (booster effect) to the thawed or reconstituted culture when inoculated into a medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercially available frozen or dried (e.g. freeze-dried) cultures may have lost a large portion of their metabolic activity but may still retain viability. For example, cultures may lose acidogenic (acidifying) activity when stored even for short periods of time. Thus, viability and booster effect need to be evaluated with different assays. Viability is evaluated with a viability assay, such as measuring colony forming units, whereas booster effect is evaluated by quantifying the relevant metabolic activity of the thawed or reconstituted culture compared to the viability of the culture.

[0055] The acidification activity assay described below is one example of an assay to quantify the relevant metabolic activity of a thawed or reconstituted culture. Although acidogenic activity is exemplified here, the present invention is intended to encompass the stabilization of any type of metabolic activity of a culture. Thus, the term "metabolic activity" refers to the oxygen scavenging activity of a culture, its acidogenic activity, i.e., the production of, for example, lactic acid, acetic acid, formic acid and / or propionic acid, or its metabolite-producing activity, such as the production of aroma compounds such as acetaldehyde, (α-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0056] According to one embodiment of the invention, the frozen culture comprises 0.2% to 20% of the cryoprotectant or drug mixture measured as a weight % of the frozen material. However, it is preferred to add the cryoprotectant or drug mixture in an amount ranging from 0.2% to 15% by weight, more preferably 0.2% to 10%, more preferably 0.5% to 7%, even more preferably 1% to 6%, including 2% to 5% of the cryoprotectant or drug mixture measured as a weight % of the frozen material. According to a preferred embodiment, the culture comprises about 3% of the cryoprotectant or drug mixture measured as a weight % of the frozen material. The preferred amount of cryoprotectant, about 3%, corresponds to a concentration in the range of 100 mM. It is noted that according to each aspect of the embodiment of the invention, the ranges may be increments of the stated ranges.

[0057] If the culture is a dried culture (e.g. freeze-dried), the cryoprotectant or mixture of agents is preferably added in an amount within the range of 0.8% to 60%, or within the range of 0.8% to 55%, or within the range of 1.3% to 40%, or within the range of 3% to 30%, or within the range of 6% to 25% (including the range of 10% to 24% of the weight of the dried culture) of the dried culture. According to a preferred embodiment, the dried culture (e.g. freeze-dried) comprises about 16% of the cryoprotectant or mixture of agents measured as a % by weight of the dried culture.

[0058] In addition, the frozen or dried culture may further contain conventional additives, including nutrients such as yeast extract, sugars, antioxidants, inert gases, vitamins, etc. Surfactants, including Tween® compounds, may also be used as further additives to the culture of the present invention. Further examples of conventional additives that can be added to the culture of the present invention include proteins, protein hydrolysates, amino acids, etc. Preferred suitable examples of these include those selected from the group consisting of glutamic acid, lysine, sodium glutamate, sodium caseinate, malt extract, skimmed milk powder, whey powder, yeast extract, gluten, collagen, gelatin, elastin, keratin, and albumin, or mixtures thereof.

[0059] More preferred conventional additives are carbohydrates.Suitable examples of these include those selected from the group consisting of pentose (e.g., ribose, xylose), hexose (e.g., fructose, mannose, sorbose), disaccharides (e.g., sucrose, trehalose, melibiose, lactulose), oligosaccharides (e.g., raffinose), oligofructose (e.g., actilite, fribrulose), polysaccharides (e.g., maltodextrin, xanthan gum, pectin, alginate, microcrystalline cellulose, dextran, PEG), and sugar alcohols (sorbitol, mannitol, and inositol).

[0060] The ratio (wt % / wt %) of the at least one cryoprotectant to the concentrated microbial culture or concentrated lactic acid bacteria culture is preferably within the range of 1:0.5 to 1:5, for example 1:1 to 1:4, or 1:1.5 to 1:3.

[0061] Another embodiment of the present invention is a method for preparing a microbial culture in high yield as described herein, further comprising drying the concentrated microbial culture or concentrated lactic acid bacterial 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.

[0062] The at least one heme protein may be present in the culture medium or may be added to the culture medium before the at least one microbial strain and / or lactic acid bacteria is added to the medium, or the at least one heme protein may be added immediately after the at least one microbial strain and / or lactic acid bacteria is added to the culture medium.

[0063] According to one embodiment, the at least one hemoprotein is a protein having natural biological catalytic activity. According to a preferred embodiment, the hemoprotein is an enzyme such as catalase or peroxidase. According to a particularly preferred embodiment, the hemoprotein is a catalase, such as, but not limited to, Catazyme®.

[0064] According to one embodiment, at least one heme protein having a native biological catalytic activity is inactivated. To achieve the goal of inactivating the native biological catalytic activity, several inactivation methods can be used, such as pH (base) inactivation, enzymatic digestion, or heat inactivation. According to a preferred embodiment, the inactivation is heat inactivation. Heat inactivation can be performed by any method known in the art, such as, but not limited to, autoclaving and / or UHT.

[0065] The inventors have surprisingly found that the addition of a thermostabilizing compound allows industrial processing of hemoproteins while at the same time allowing high growth yields. According to one embodiment, the culture medium further comprises a thermostabilizing compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers, and non-ionic detergents. The thermostabilizing compound is 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 (types A and B), hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, fucoidan, pentosan polysulfate, 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, polyacrylic 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). According to a preferred embodiment, the thermostabilizing compound is sorbitol.

[0066] According to one embodiment, the hemoprotein is a protein that has no inherent biological activity.

[0067] According to one embodiment, the heme protein is produced by a microorganism. According to one embodiment, the heme protein is indirectly derived from or directly produced by Aspergillus niger. According to one embodiment, the heme protein is indirectly derived from or directly produced by Pichia pastoris. According to one embodiment, the heme protein is indirectly derived from or directly produced by Escherichia coli. According to one embodiment, the heme protein is indirectly derived from or directly produced by Bacillus.

[0068] At least one heme protein is added to or present in the culture medium as a feedstock to aid in fermentation. The inventors have surprisingly discovered that application of a non-plant derived source to the culture medium can replace the at least one heme protein without loss of yield.

[0069] Thus, one aspect of the present invention relates to the use of at least one heme protein in a fermentation method and / or process.

[0070] The culture medium may be a complex fermentation medium.

[0071] The complex fermentation medium can be any complex fermentation medium known in the art, but the complex fermentation medium can include compounds selected from the group consisting of lactose, nutrients, vitamins, tryptone, soy peptone, yeast extract, ascorbic acid, magnesium sulfate, milk, and combinations thereof.

[0072] According to one embodiment, the heme protein is added at a level that allows respiration in excess of the natural oxygen consumption that the cells can support. The heme protein stimulates aerobic microbial growth in a dose-dependent manner, with oxygen consumption, as a measure of microbial growth, peaking earlier and at a faster rate compared to cultures without the heme protein.

[0073] Thus, according to one embodiment, oxygen consumption during the fermentation reaches a maximum in less than 12 hours, such as less than 10 hours or less than 8 hours.

[0074] According to one embodiment, the oxygen consumption during the fermentation reaches 0.04 mol O2 / L / h in less than 10 hours or within 8 hours.

[0075] Oxygen consumption can be measured using any method known to one of skill in the art.

[0076] According to one embodiment the culture medium in step (i) comprises at least 0.5% w / w of at least one heme protein, such as 1% w / w, for example 2% w / w, such as 3% w / w, for example 4% w / w, such as 0.5-4% w / w, for example 1-3.5% w / w, such as 1.5-3% w / w, for example 2-2.5% w / w of at least one heme protein strain, based on the weight of the culture medium, prior to fermentation (i.e. before the at least one microbial strain is added).

[0077] According to a further embodiment, the culture medium in step (i) comprises at least 0.5% w / w of a microbial inoculum culture, such as a pre-fermented lactic acid bacteria inoculum culture, such as at least 1% w / w, such as 1.5% w / w, 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, such as in the range of 0.5-4% w / w, such as 1-3.5% w / w, for example 1.5-3% w / w, such as 2-2.5% w / w of a pre-fermented lactic acid bacteria inoculum culture relative to the weight of the culture medium (i.e. before the at least one microbial strain is added).

[0078] According to a preferred embodiment, the heme protein is added at a concentration of about 0.1 g per kg of fermentate to about 10 g per kg of fermentate.

[0079] Surprisingly, by the method of the invention it is sometimes 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 applying the method of the invention, most cultures need to be concentrated in order to obtain a commercially valuable starter culture. Such cultures can preferably be harvested and concentrated by centrifugation or ultrafiltration.

[0080] Furthermore, according to a preferred embodiment, the cultivation is carried out in a large-scale fermenter containing 5 L to 100,000 L of culture medium, preferably 300 L to 20,000 L of culture medium.

[0081] In a preferred embodiment, the culture includes temperature and / or pH control.

[0082] According to one embodiment, the culture medium of step (i) and / or step (ii) comprises one or more microbial strains which are microbial strains incapable of respiratory growth without supplementation with components / alternative components of the respiratory chain.

[0083] According to one embodiment, the culture medium in step (i) and / or step (ii) comprises at least one microbial strain selected from the following group: Lactococcus, Streptococcus, Lactobacillus now known as Lactobacillus, Holzapfelia, mylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus (Lactococcus spp.), Lactobacillus spp., ... Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus (Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc., Oenococcus., Leuconostoc.), Pediococcus, Enterococcus, Bifidobacterium, Brevibacterium, Propionibacterium and combinations thereof.

[0084] According to one embodiment, the culture medium in step (i) and / or step (ii) comprises at least one lactic acid bacterium selected from the following group: Lactococcus, Streptococcus, Lactobacillus now known as Lactobacillus, Holzapfelia, mylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Lactic acid bacterium ... acticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus (Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107), Leuconostoc., Oenococcus., Leuconostoc.), Pediococcus, Enterococcus, and Bifidobacterium.

[0085] According to one embodiment, the culture medium in step (i) and / or step (ii) comprises one or more mesophilic bacteria selected from the following group: Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei (new name Lactobacillus casei subsp. casei) (Lacticaseibacillus casei), Lactobacillus casei. paracasei (Lactobacillus paracasei subsp. paracasei) (Lacticaseibacillus paracasei subsp. paracasei and Lacticaseibacillus paracasei subsp. tolerans) and Oenococcus oeni.

[0086] According to a further embodiment, 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.

[0087] According to a further embodiment, the culture medium in step (i) and / or step (ii) comprises one or more thermophilic bacteria selected from the group consisting of Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus.

[0088] According to one embodiment, the culture medium in step (i) and / or step (ii) is an LD-culture comprising one or more organisms selected from the following group: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis, and Leuconostoc mesenteroides subsp. cremoris. The term "LD culture" here should be understood as a combination of the species Lactococcus lactis and Leuconostoc.

[0089] It is understood 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 the present specification, "O-culture" is understood as a culture medium comprising Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. O-cultures are usually used for the production of cheeses without holes (Cheddar, Cheshire, Feta). This particular culture is commercially available under the name R604 from Chr. Hansen A / S, Denmark (catalog number 200113).

[0090] According to a preferred embodiment, the culture medium in step (i) and / or step (ii) is a culture comprising Lactococcus lactis.

[0091] To obtain maximum yield, the harvest in step (ii) is preferably carried out 5 hours and 24 hours after the start of the culture.

[0092] The method of the present invention may further comprise storing the harvested microbial or lactic acid bacteria culture obtained in step (ii) or the concentrated microbial or lactic acid bacteria culture obtained in step (iii).

[0093] Due to the high yield of this method, the microbial culture in the fermentation obtained in step (i) can contain in the range of 2.0E+10 to 5.0E+10 active microbial cells per gram of microbial culture, for example, 2.5E+10 to 4.5E+10, for example, 3.0E+10 to 4.0E+10 active microbial cells. Similarly, the microbial culture in the fermentation obtained in step (i) can contain in the range of 2.0E+10 to 5.0E+10 total microbial cells per gram of microbial culture, for example, 2.5E+10 to 4.5E+10, for example, 3.0E+10 to 4.0E+10 total microbial cells. In Table 2 of the experimental part, it can be seen that 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 the lactic acid bacteria starter culture obtained by the present invention have high viability.

[0094] Due to the high yield of this method, the lactic acid bacteria culture in the fermentation product obtained in step (i) can contain in the range of 2.0E+10 to 5.0E+11 active lactic acid bacteria cells per gram of lactic acid bacteria culture, for example, 2.5E+10 to 4.5E+10, for example, 3.0E+10 to 4.0E+10 active lactic acid bacteria cells. Similarly, the lactic acid bacteria culture in the fermentation product obtained in step (i) can contain in the range of 2.0E+10 to 5.0E+10 total lactic acid bacteria cells per gram of lactic acid bacteria culture, for example, 2.5E+10 to 4.5E+10, for example, 3.0E+10 to 4.0E+10 total lactic acid bacteria cells. In Table 2 of the experimental part, it can be seen that 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 the lactic acid bacteria starter culture obtained by the present invention have high viability.

[0095] Viable cell numbers and / or total cell numbers are determined using flow cytometry, a technique known to those of skill in the art.

[0096] According to a preferred embodiment, said increased yield of the harvested microbial strain, e.g. lactic acid bacteria or microbial culture, e.g. lactic acid bacteria culture, of this method is increased by 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 the anaerobic process excluding the heme source process.

[0097] According to a second aspect of the invention, the invention relates to a microbial culture, such as a starter culture, obtainable by the method of the first aspect of the invention. The microbial culture, such as a starter culture, may be provided as a culture concentrate, such as a starter culture concentrate.

[0098] According to a third aspect, the invention relates to a microbial culture, such as a starter culture, comprising at least one heme protein.

[0099] A fourth aspect relates to a culture medium comprising at least one heme protein.

[0100] A fifth aspect of the invention relates to a method of preparing food, feed, pharmaceutical, dairy flavouring and cheese flavouring products, said method comprising adding an effective amount of a culture according to the second or third aspect to a food, feed or pharmaceutical starting material and maintaining the inoculated culture under conditions in which at least one microbial strain is metabolically active.

[0101] Preferably the food product of the fifth aspect of the invention is selected from the group consisting of dairy products, vegetable products, meat products, beverages, fruit juices, wine, bakery products, dairy flavourings and cheese flavouring products.

[0102] Preferably, the dairy product is selected from the group consisting of cheese, yoghurt, butter, inoculated sweet milk and liquid fermented dairy products.

[0103] According to a sixth aspect, the present invention relates to a fermented food, feed or pharmaceutical product obtainable by the method of the first aspect.

[0104] A seventh aspect of the invention relates to the use of at least one heme protein in a fermentation method and / or process.

[0105] Compounds produced by the described microorganisms include, but are not limited to, enzymes, proteins, metabolites, glycolipids, antibiotics, bacteriocins, amino acids, flavors, volatiles, etc. Such compounds can be produced by recombinant DNA technology or conventional methods.

[0106] An eighth aspect relates to a food, feed product, pharmaceutical, dairy flavour or cheese flavour product comprising a culture according to the second or third aspect. EXAMPLES

[0107] Example 1: Fermentation was carried out using catalase as a hemoprotein in a complex fermentation medium from Chr. Hansen A / S Stock solutions of catalase (Catazyme®, purchased from NovoZymes) at concentrations between 24% and 55% (w / w) are prepared in water. The catalase solution is then treated at ultra-high temperature (UHT) at 141 °C for 8–10 s to sterilize the material and completely inactivate the enzyme. The Catazyme® product contains 9% w / w catalase and 42% w / w sorbitol. Surprisingly, sorbitol was found to act as a protein stabilizer. When Catazyme® solutions are UHT treated at concentrations below 24% w / w, the heme molecules are decomposed at high temperatures and are no longer able to support L. lactis respiration (data not shown). Thus, sorbitol acts as a protein stabilizer and heme protectant.

[0108] culture The experiments were performed using a commercially available Lactococcus lactis culture deposited on March 15, 2011 by Chr. Hansen A / S, Horsholm, Denmark at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany under the accession number DSM 24648.

[0109] Fermentation medium The fermentation medium used was a proprietary vegetarian complex fermentation medium from Chr. Hansen A / S.

[0110] Negative control As a negative control medium, an anaerobic complex fermentation medium was used, which was a proprietary vegetarian complex fermentation medium of Chr. Hansen A / S without a heme source.

[0111] Positive control The positive control medium was an aerobic complex fermentation medium, a proprietary vegetarian complex fermentation medium from Chr. Hansen A / S, containing a non-vegetarian heme source.

[0112] The medium was sterilized by UHT treatment (141°C for 8-10 seconds). The pH of the finished medium was 6.5.

[0113] Fermentation conditions of the culture Fermentations were carried out in 2L lab-scale fermentation tanks at 30°C with aeration using 1% (w / w) of the above culture as inoculum and one of the above heme proteins Catazyme® as heme source. For the aerobic fermentation as positive control, the same conditions as for the aerobic fermentation were applied with aeration using Chr. Hansen A / S's proprietary vegetarian complex fermentation medium containing a non-vegetarian heme source. For the anaerobic fermentation as negative control, the same conditions as for the aerobic fermentation were applied but without aeration using Chr. Hansen A / S's proprietary vegetarian complex fermentation medium excluding the heme source. The culture was acidified to pH 6.0. The pH was then maintained at 6.0 by controlled addition of 27% NH4OH.

[0114] Once no further consumption of base was detected, each culture was cooled to approximately 10°C.

[0115] After cooling, the bacteria in the culture medium were concentrated 6–18-fold by centrifugation and then frozen as pellets in liquid nitrogen at 1 atm, generating so-called frozen direct vat set cultures (F-DVS). The F-DVS pellets were stored at −50°C until further analysis.

[0116] Process Parameter Evaluation Lactococcus lactis undergoes significant metabolic changes when transitioning from anaerobic to respiratory growth. Compared to anaerobic growth, biomass approximately doubles during respiratory growth and acid production decreases. An important feature of respiratory growth is the decrease in dissolved oxygen (DO%) (Figure 1). Compared to the aerobic positive control, the respiratory fermentation process using Catazyme® (heme protein, 9% w / w) showed similar dissolved oxygen (DO%).

[0117] In conclusion, based on the process curves in Figure 1, the respiratory fermentation process using Catazyme® as the heme protein performed comparable to the aerobic positive control (Figure 1, non-vegetarian heme source) in supporting respiratory growth.

[0118] Downstream process evaluation After fermentation, the fermentate is subjected to a Packed Cell Volume (PCV) test (the fermentate is centrifuged in special centrifuge tubes), which is the first indicator of biomass. The PCV test showed that fermentation with Catazyme® gave a PCV with almost the same levels of bacterial cells as the aerobic positive control process, whereas the anaerobic negative control had lower PCV levels (data not shown).

Claims

1. The following steps: (i) culturing at least one microbial strain in a culture medium under aeration to obtain a fermentate, wherein said culture medium contains at least one heme protein; (ii) harvesting the microbial strain from the fermentation to obtain a microbial culture; A method for obtaining a microbial culture comprising:

2. The method of claim 1 , wherein the hemoprotein is a protein having native biological catalytic activity.

3. The method of claim 2 , wherein the hemoprotein is an enzyme.

4. 2. The method of claim 1, wherein the hemoprotein is a protein that has no native biological catalytic activity.

5. The method according to any one of claims 1 to 4, wherein the oxygen consumption in the fermentation broth reaches 0.04 mol O2 / L / h in less than 10 hours or within 8 hours.

6. The method according to any one of claims 1 to 5, wherein the hemoprotein is produced by a microorganism.

7. 7. The method of any one of claims 1 to 6, wherein the hemoprotein is produced by expression from Aspergillus, Pichia, Bacillus, or Escherichia.

8. The method of claim 7, wherein the hemoprotein is catalase or peroxidase.

9. 9. The method of claim 1, wherein the hemoprotein is added at a concentration of about 0.1 g to about 10 g per kg of fermentate.

10. The method of any one of claims 1 to 9, further comprising the step of (iii) concentrating the microbial culture to obtain an enriched microbial culture.

11. The method of any one of claims 1 to 10, further comprising the step of (iv) freezing or drying the microbial culture to obtain a frozen or dried microbial culture.

12. 12. The method of claim 10 or 11, further comprising the step of (v) packaging the frozen microbial culture or the dried microbial culture obtained in step (iv).

13. The method of claim 2 or 3, wherein the hemoprotein is an inactivated hemoprotein.

14. The method of claim 13 , wherein the heat-inactivated hemoprotein is heat-inactivated.

15. The method according to any one of claims 1 to 14, wherein the culture medium is free of non-vegetarian hemoproteins.

16. The method of any one of claims 1 to 15, wherein the hemoprotein is a catalase enzyme, such as Catazyme®.

17. The method of any one of claims 1 to 16, wherein the culture medium further comprises a thermostabilizing compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers and non-ionic surfactants.

18. The heat stabilizing compound is 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 (types A and B), hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, fucoidan, pentosan polysulfate, 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, polyacrylic 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), the method of claim 17.

19. A culture obtained by the method according to any one of claims 1 to 18. A culture obtainable by the method according to any one of claims 1 to 18.

20. A culture or culture medium comprising at least one heme protein.

21. 21. The culture or culture medium of claim 20, wherein the heme protein is catalase.

22. 22. The culture or culture medium of claim 21, further comprising a thermostabilizing compound selected from the group consisting of polyols, sugars, biopolymers, amino acids, salts, polymers and non-ionic surfactants.

23. The heat stabilizing compound is 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 (types A and B), hydroxyethyl starch, poly-L-glutamic acid, poly-L-lysine, fucoidan, pentosan polysulfate, keratan sulfate, polyaspartic acid, polyglutamic acid, hydroxyethyl cellulose, hydroxypropyl-β-cyclodextrin, glycine, L-alanine, glycine, L-glycerol, ... arginine hydrochloride, arginine, proline, lysine, histidine, aspartic acid, glutamic acid, acetate, citrate, sodium chloride, phosphate, ascorbate, polyacrylic 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), the culture or culture medium of claim 22.

24. 24. A method for preparing food, feed, pharmaceutical, dairy flavouring and cheese flavour products, comprising adding an effective amount of the culture of any one of claims 19 to 23 to a food, feed or pharmaceutical starting material and maintaining the inoculated culture under conditions in which at least one microbial strain is metabolically active.

25. A fermented food, feed or pharmaceutical product obtained by the method according to any one of claims 1 to 18.

26. Use of at least one heme protein in a fermentation method and / or process.

27. A food, feed, pharmaceutical, dairy flavour or cheese flavour product comprising the culture of any one of claims 19 to 23.

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