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Title:
FOOD, FOOD PRECURSORS OR BEVERAGES COMPRISING D-LACTIC ACID AND/OR A SALT THEREOF AND A METHOD OF PRODUCING THE SAME
Document Type and Number:
WIPO Patent Application WO/2021/032896
Kind Code:
A1
Abstract:
The present invention relates to food, food precursors or beverages comprising D-lactic acid and/or a salt thereof and a method for producing the same. Further, the present invention relates to the use of lactic acid bacteria in a method for producing food, food precursors or beverages comprising D-lactic acid and/or a salt thereof and to lactic acid bacteria strains for use in this method. The present invention is based on the finding that D-lactic acid and/or a salt thereof can protect mitochondria against intrinsic (e.g. genetic mutation) or extrinsic (e.g. mitochondrial toxins, pesticides) factors that lead to mitochondrial dysfunction, whereas L-lactic acid is not capable to support, maintain or reestablish mitochondrial function.

Inventors:
FRIEDRICHSON TIM (DE)
FESTEL GUNTER (CH)
Application Number:
PCT/EP2020/073666
Publication Date:
February 25, 2021
Filing Date:
August 24, 2020
Export Citation:
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Assignee:
BRAINBOOST SOLUTIONS (CH)
International Classes:
A23L21/10; A23L29/00; A23L31/00; C12P7/56
Domestic Patent References:
WO2017135364A12017-08-10
WO2010143323A12010-12-16
WO2015150383A12015-10-08
WO1995022911A11995-08-31
Foreign References:
US5520936A1996-05-28
US8535742B22013-09-17
KR20160101660A2016-08-25
CN103114053A2013-05-22
CN109259203A2019-01-25
EP2596703A12013-05-29
JP2015080429A2015-04-27
Other References:
ZHANG YIXING ET AL: "d-Lactic acid biosynthesis from biomass-derived sugars viaLactobacillus delbrueckiifermentation", BIOPROCESS AND BIOSYSTEMS ENGINEERING, SPRINGER, DE, vol. 36, no. 12, 14 May 2013 (2013-05-14), pages 1897 - 1904, XP035365272, ISSN: 1615-7591, [retrieved on 20130514], DOI: 10.1007/S00449-013-0965-8
BUENAVENTURADA P CALABIA ET AL: "Production of d-lactic acid from sugarcane molasses, sugarcane juice and sugar beet juice by Lactobacillus delbrueckii", BIOTECHNOLOGY LETTERS, SPRINGER NETHERLANDS, DORDRECHT, vol. 29, no. 9, 31 May 2007 (2007-05-31), pages 1329 - 1332, XP019523948, ISSN: 1573-6776, DOI: 10.1007/S10529-007-9408-4
NGUYEN CUONG MAI ET AL: "d- andl-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation", BIOCHEMICAL ENGINEERING JOURNAL, ELSEVIER, AMSTERDAM, NL, vol. 81, 12 October 2013 (2013-10-12), pages 40 - 46, XP028780574, ISSN: 1369-703X, DOI: 10.1016/J.BEJ.2013.10.003
KLOTZ SILVIA ET AL: "Biotechnological production of enantiomerically pured-lactic acid", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 100, no. 22, 22 September 2016 (2016-09-22), pages 9423 - 9437, XP036081195, ISSN: 0175-7598, [retrieved on 20160922], DOI: 10.1007/S00253-016-7843-7
YÁÑEZ R ET AL: "Production of D(-)-lacticacid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subs. torquens", BIOTECHNOLOGY LETTERS, KLUWER ACADEMIC PUBLISHERS, DORDRECHT, vol. 25, no. 14, 1 July 2003 (2003-07-01), pages 1161 - 1164, XP002701476, ISSN: 0141-5492, DOI: 10.1023/A:1024534106483
CHACKO ET AL., CLIN. SCI., vol. 127, 2014, pages 367 - 373
Attorney, Agent or Firm:
STOLMÁR & PARTNER PATENTANWÄLTE PARTG MBB (DE)
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Claims:
Claims

1. A food product comprising lactate, wherein at least 80% of the lactate is D-lactate.

2. The food product according to claim 1 for use in the prevention and treatment of disorders associated with decreased mitochondrial function, in particular cancer, cardiovascular diseases, chronic fatigue syndrome, chronic infections, diabetes and metabolic syndrome, neurobehavioral and psychiatric diseases, such as autism spectrum disorders, schizophrenia, and bipolar and mood disorders, neurodevelopmental disorders or skeletal muscle hypertrophy and atrophy.

3. The food product according to claim 1 or 2, additionally comprising one or more selected from micronutrients, functional foods, food supplements or other compounds.

4. The food product according to any of claims 1 to 3, wherein the food product is in a liquid, semiliquid or solid form.

5. The food product according to any of claims 1 to 4, wherein the food product is a fruit- based product.

6. The food product according to any one of claims 1 to 4, wherein the total amount of D-lactate contained in the food product is at least 0.8 % (w/w), preferably 2 % (w/w) up to 6 % (w/w).

7. Method for producing a food product comprising lactate, wherein at least 80% of the lactate is D-lactate, said method comprising the steps of: a) providing a first substrate; b) treating the first substrate with microorganisms.

8. The method according to claim 7, wherein the first substrate is plants or parts of plants, fungi, algae, molasses, botanicals or processed products thereof. 9. The method according to claim 7 or 8, wherein the microorganism is a lactic acid bacterium selected from the group of Lactobacillus coryniformis, Lactobacillus jensenii, Lactobacillus delbrueckii, Sporolactobacillus inulinus.

10. The method according to any of claims 7 to 9, wherein the method further comprises the steps of: c) providing a second substrate, and d) treating the second substrate with microorganisms; and e) mixing the first substrate obtained in step (b) with the second substrate,

11. The method according to any of claims 7 to 10, wherein the food product obtained by the method is ready for consumption.

12. The method according to any of claims 7 to 11 , wherein the method further comprises the step of processing the substrate obtained in steps (b) and/or (d) to a beverage.

13. Food product produced by a method according to any one of claims 7 to 12.

14. Use of lactic acid bacteria in a method for producing a food product comprising lactate, wherein at least 80% of the lactate is D-lactate.

15. Lactic acid bacteria strain for use in a method for producing a food product comprising lactate, wherein at least 80% of the lactate is D-lactate and wherein the amount of D-lactate is at least 0.8 % (w/w), preferably 2 % (w/w) up to 6 % (w/w).

Description:
Food, food precursors or beverages comprising D-lactic acid and/or a salt thereof and a method of producing the same

The present invention relates to food products comprising D-lactate and methods for producing the same. Further, the present invention relates to the use of these food products in the treatment and preventions of disorders associated with decreased mitochondrial stability.

Lactic acid is a chiral alpha-hydroxy acid consisting of two optical isomers, L-(+)-lactic acid or (S)-lactic acid and D-(-)-lactic acid or (R)-lactic acid. Both isomers differ in their metabolism, tissue and plasma concentration and biological function. While L-lactic acid historically has been regarded as the desirable, physiologically active and metabolic energy providing isomer, D-lactic acid has long been considered to be metabolically inert.

Lactic acid has the lUPAC name 2-hydroxypropanoic acid and the chemical formula C3H6O3. Lactic acid is found primarily in sour milk products, such as koumiss, laban, yogurt, buttermilk, kefir, some cottage cheeses and kombucham, but also in pickled vegetables, cured meats and fish. Both optical isomers of lactic occur naturally in fermented foods. For example, WO 95/22911 discloses an alcohol-free refreshing drink produced organically from pure natural products.

Lactic acid is widely used in plastic industry, food and drug industry, and cosmetic industry. Most of the industrial production of lactic acid is achieved by fermentation of carbohydrates such as glucose, sucrose, maltose, starch or cellulose using microorganisms capable of producing lactic acid such as bacteria and fungi. At present, L- lactic acid is widely produced in an industrial scale.

Lactic acid and salts thereof are approved food additives in the EU, US, Australia, New Zealand and Japan. Lactic acid is furthermore listed by its INS number 270 or as E number E270. Lactic acid is used in the art as a food preservative, curing agent, and flavoring agent. It is used in processed foods and as a decontaminant during meat processing. The amount of L- and D-lactic acid that can be added to food products is generally without limitations, except for those imposed by good manufacturing practice.

The regular amount of lactic acid is, for example, for yoghurt about 0.8% (w/w), and for buttermilk 1.5% (w/w). However, the majority of lactic acid contained in these products is L-lactic acid. In general, known food products contain only a low content of D-lactic acid. However, it has been recently described that D-lactic acid can protect mitochondria against intrinsic (e.g. genetic mutation) or extrinsic (e.g. mitochondrial toxins, pesticides) factors that lead to mitochondrial dysfunction. Surprisingly, L-lactic acid was not capable to support, maintain or reestablish mitochondrial function.

The objective of the present invention is therefore to provide food products comprising a high content of D-lactic acid and/or a salt thereof and a method for producing the same.

To this end, the present inventors have established conditions suitable for producing food products containing high concentrations of D-lactic acid by fermentation using D-lactic acid producing bacteria. Hence, the food products of the invention comprise more D-lactic acid and/or a salt thereof than the amount of D-lactic acid found in natural food.

In a first aspect, the invention therefore relates to food products comprising lactate, wherein at least 80% of the lactate is D-lactate.

Food products are herein meant to encompass not only finished, marketable food products, but also food precursors, ingredients for food products and beverages. Food products can be plant or animal based. In one aspect of the invention, the food products are plant based. In a preferred embodiment, the food products are fruit based. Food products and their precursors have to fulfill certain quality criteria and be approved for human consumption. The same holds true for the ingredients used to produce such food products. Consequently, laboratory by-products are no food products because they have been produced without being intended or suitable for human consumption.

The food products according to the invention comprise lactate. Lactate as used herein refers to both lactic acid as well as salts thereof. Consequently, the term “D-lactate” as used herein refers to D-lactic acid as well as salts thereof.

The lactate comprised in the food products according to the invention is at least 80% D- lactate. That means that only 20% of the lactate contained in the food products according to the invention is L-lactate. This is contrast to food products known in the art such as yoghurt, which comprise mostly L-lactate.

In one embodiment, at least 90% or 95% of the lactate contained in the food products of the invention is D-lactate. Because the content in D-lactate in the food products according to the invention is very high, these food products provide an amount of D-lactate that is sufficient for supporting, maintaining and/or reestablishing mitochondrial function.

Mitochondrial function or activity as used herein is defined as the mitochondria’s ability to consume oxygen and produce energy in the form of adenosine-triphosphate (ATP). Mitochondrial function can decline due to ageing, toxin exposure or in the course of specific diseases. Reduced mitochondrial function is herein defined as reduced mitochondrial membrane potential, defects in ATP synthesis, perturbed network structure and increased generation of reactive oxygen species, among other aspects. Disorders associated with decreased mitochondrial function include cancer, cardiovascular diseases, chronic fatigue syndrome, chronic infections, diabetes and metabolic syndrome, neurobehavioral and psychiatric diseases, such as autism spectrum disorders, schizophrenia, and bipolar and mood disorders, neurodevelopmental disorders or skeletal muscle hypertrophy and atrophy. Further disorders associated with a decreased mitochondrial function are autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes; gastrointestinal disorders; musculoskeletal diseases, such as fibromyalgia. Mitochondrial function can be measured by means known in the art, for example as bioenergetic health index.

Because of their high content in D-lactate, the food products according to the invention are useful in the treatment or prevention of disorders associated with decreased mitochondrial function, in particular cancer, cardiovascular diseases, chronic fatigue syndrome, chronic infections, diabetes and metabolic syndrome, neurobehavioral and psychiatric diseases, such as autism spectrum disorders, schizophrenia, and bipolar and mood disorders, neurodevelopmental disorders or skeletal muscle hypertrophy and atrophy.

In one embodiment, the food products comprise a total amount of D-lactate of at least 0.8 % (w/w), preferably 2 % (w/w) up to 6 % (w/w).

In another embodiment, the food products further comprise one or more selected from micronutrients, functional foods, food supplements or other compounds.

Micronutrients are essential elements required in small quantities throughout the life cycle to orchestrate a range of physiological functions to maintain health. For human nutrition, micronutrient requirements are in amounts generally less than 100 milligrams per day. Micronutrients are vitamins, minerals (quantity elements and trace elements), proteinogenic amino acids and omega fatty acids, for example Vitamin A, Bi, B 2 , B 3 , Bb, Bi2, C, D, E, niacin, coenzyme Q10, iodine, iron and selenium.

In a preferred embodiment, the food product according to the invention comprises micronutrients selected from short-chain fatty acids, polyphenols (anthocyanins), trace minerals and vitamin D.

Functional food is food enriched with additional ingredients, for example vitamins, minerals, bacterial cultures and unsaturated fatty acids.

Food supplements are dietary supplements comprising concentrates of nutrients or other substances having a nutritional or physiological effect alone or in combination, which are intended to supplement the diet.

The food products according to the invention may be in a liquid, semiliquid or solid form. In a preferred embodiment, the food product according to the invention is a beverage.

Food products according to the present invention may comprise a total content of D- lactate of at least 0.8 % (w/w), preferably 2 % (w/w) up to 6 % (w/w) and one or more selected from micronutrients, functional foods, food supplements or other compounds.

In a second aspect, the invention relates to a method for producing a food product comprising lactate, wherein at least 80% of the lactate is D-lactate, said method comprising the steps of: a) providing a first substrate; b) treating the first substrate with microorganisms.

The method according to the invention may additionally comprise the steps of: c) providing a second substrate; d) treating the second substrate with microorganisms; and e) mixing the first substrate obtained in step (b) with the second substrate obtained in step (d).

In accordance with a preferred embodiment of the invention, the first and/or second substrates may comprise plants or parts thereof, fungi, algae, molasses, botanicals, in particular all monosaccharide-containing botanicals commonly used in human and animal nutrition, and processed products of the above-mentioned substrates, comprising juices, pulps, extracts and blends thereof. In a preferred embodiment, the substrate is fruit- based.

In one embodiment, the carbohydrates contained in the first and/or second substrate can be first enzymatically hydrolysed, for example by incubating the substrate with amylase, cellulase, pectinase, peptidase or xylanase. This increases the concentration of readily fermentable monosaccharides.

In a preferred embodiment of the invention, the microorganism used for treating the first and/or second substrate is a lactic acid bacterium that completely or predominantly produces D-lactic acid. Homofermentative lactic acid bacteria strains, such as Lactobacillus, Streptococcus, Enterococcus, Lactococcus, Pediococcus, are preferred because of their ability to convert hexoses (e.g., glucose) as a substrate into two lactic acid molecules without byproducts. In contrary, heterofermentative lactic acid bacteria strains, for example Leuconostoc, convert pentoses or hexoses to lactic acid and byproducts, such as ethanol and acetic acid.

In a more preferred embodiment, the lactic acid bacterium is selected from the group of Lactobacillus coryniformis, Lactobacillus jensenii, Lactobacillus delbrueckii, Sporolactobacillus inulinus. All of these lactic acid bacteria strains are known to produce mostly D-lactate.

Treating the first and/or second substrate with microorganism according to the present invention comprises fermentation, i.e. the microbial or enzymatic conversion of organic substances into (lactic) acid. Treating the first substrate may comprise anaerobic fermentation, aerobic/oxidative fermentation, conversion of organic materials due to microbial or enzymatic processes with or without involvement of oxygen (bioconversion and biocatalysis), controlled production of biotic metabolites with or without involvement of oxygen, submersfermentation, solid phase-fermentation on a carrier material, batch fermentation, fed-batch fermentation or continuous fermentation.

Mixing of the first substrate obtained in step (b) with the second substrate obtained in step (d) may comprise mixing, stirring or kneading.

In one embodiment, steps c) and d) can be repeated during the fermentation. Substrates differing from each other can be added.

In a further embodiment, substrates are added continuously and final products are removed continuously.

In another embodiment of the present invention, the method comprises first mixing a first and a second substrate and then treating the mixture with microorganisms.

In another embodiment of the present invention, the method comprises the step of mixing a second substrate with the first treated substrate and then treating the mixed substrate.

In a preferred embodiment, the food product obtained by the method is ready for consumption. That means that no further steps such as refining or purification are necessary to bring the food product in a form that can be approved for human consumption.

In another embodiment, the method according to the present invention further comprises the step of processing the substrate obtained in steps (b) and/or (d) to a beverage.

Processing of the substrate obtained in steps (b) and/or (d) to a beverage may comprise mixing, stirring, mashing, crushing, chopping, squeezing or extracting.

The product obtained in step b), d) or e) may remain untreated and be directly administered as a food product. In another embodiment, the microorganisms used are killed by methods known in the art after step b), d) or e). In a preferred embodiment, the microorganism is killed by applying heat after step b), d) or e), preferably by pasteurization with subsequent cooling. The method according to the invention may further comprise the steps of direct addition of synthetic D-lactic acid and/or semi-synthetic D-lactic acid to the first and/or second substrate and/or to the substrates obtained in step c), d) and e).

Direct addition of D-lactic acid and/or semi-synthetic D-lactic acid to the first and/or second substrate or to the substrates obtained in step c), d) and e) comprises addition by mixing, stirring, kneading or other means of common techniques applied to production of food, food precursors and beverages.

Further, the present invention provides the use of lactic acid bacteria in a method for producing food, food precursors or beverages comprising D-lactic acid and/or a salt thereof, preferably in a method according to the present invention.

In a preferred embodiment, the use of lactic acid bacterium comprises the use of lactic acid bacteria selected from the group of Lactobacillus coryniformis, Lactobacillus jensenii, Lactobacillus delbrueckii, Sporolactobacillus inulinus.

In another embodiment of the present invention, the method for producing food or food precursors comprising D-lactic acid and/or a salt thereof further comprises the steps of direct addition of synthetic D-lactic acid and/or semi-synthetic D-lactic acid and/or salts thereof to food, food precursors or beverages.

Addition of synthetic D-lactic acid and/or semi-synthetic D-lactic acid and/or salts thereof to food products comprises mixing, stirring, kneading or other means of common techniques applied to production of food, food precursors and beverages.

The methods according to the invention aim at providing a food product having a high content in D-lactate. This is in contrast to existing methods which were directed to provide food products containing a high content of L-lactate. Further methods described in the art are directed to the production of lactate, but not to the provision of a food product that is suitable for human consumption.

In another aspect, the present invention provides a lactic acid bacteria strain for use in a method for producing a food product comprising D-lactic acid and/or a salt thereof, wherein the amount of D-lactic acid or a salt thereof is at least 0.8 % (w/w), preferably 2 % (w/w) up to 6 % (w/w). Figures

Fig. 1 shows the enrichment of D-lactic acid by different bacterium strains and using different substrates.

Fig. 2 shows the enrichment of D-lactic acid (increase of concentration [g/L]) during fermentation over a time period of 0 to 50 h.

Fig. 3 shows the decrease of sugar concentration [g/L] during fermentation over a time period of 0 to 50 h.

Examples Example 1 - Evaluation of different bacterium strains and different substrates for the enrichment of D-lactic acid in food, food precursors or beverages

Frozen, whole mirabelles with pit were thawed completely. The mirabelles were pitted and the remaining skins and pulps were thoroughly blended. The pH of the blended mirabelle fruit masss was 3.2 Complex carbohydrates in the blended mirabelle fruit mass were optionally enzymatically hydrolyzed to increase the concentration of readily fermentable monosaccharides. Therefore, hydrolysis of mirabelle jam under use of different enzymes was performed as follows:

Substrate 1: 1 ml/kg Cellulase (Accellerase 1500) and 1.25 ml/kg Pektinase

(Pektinase L-40)

Substrate 2: 1 ml/kg Cellulase (Accellerase 1500), 1.25 ml/kg Pektinase (Pektinase L-

40) and

2.5 g/L yeast extract in powdered form

Substrate 3: 1 ml/kg Cellulase (Accellerase 1500), 1.25 ml/kg Pektinase (Pektinase L-

40) and 0.25 g/kg Protease (Fermgen) Hydrolysis was performed for 24 h at pH 4.5 (adjusted with 20% NaOH) in shake flasks at 50°C and 150 rpm. After hydrolysis, the pH was adjusted to 6.5 with 20% NaOH. A fermentation of the blended fruit mass treated as described above was performed with different D-lactic acid producing hetero- and homo-fermentative bacterial strains at their optimal temperature ranging from 30 to 52 °C as follows:

Table 1 As preculture, each bacterial strain was inoculated into Man-Rogosa-Sharpe (MRS) medium and incubated 24 h at its optimal temperature. MRS medium is a liquid medium recommended for use in the cultivation of Lactobacillus species and lactic acid bacteria.

As substrates, 10 mL each of each hydrolysed mirabelle jam (substrate 1, 2 or 3) was filled into separate sterile glass tubes. For Sporolactobacillus inulinus (DSM 20348) each hydrolysed mirabelle jam (substrate 1, 2 or 3) was additionally supplemented with 60 g/l CaCC>3. After incubation time of 24 h, 100 pL preculture containing the respective bacterial strain was added to the 10 mL mirabelle jam into the glass tube and fermentation was performed at the optimal temperature of each respective bacterial strain for 48 h.

After 48 h of fermentation, D-lactic acid and L-lactic acid concentration, respectively, was determined enzymatically with a commercial D-lactic acid (D-lactate) and L-lactic acid (L- lactate) assay kit (Megazyme, Wicklow, Ireland). Several hetero- and homo-fermentative bacterial strains produce D-lactic acid in all three substrates. However, homo-fermentative strains are preferred according to this invention, because they exclusively synthesize lactic acid and no other byproducts, such as acetate or ethanol. Control (not containing bacteria) and negative control (using strain DSM 2314, exclusively producing L-lactic acid) do not produce D-lactic acid in any of the three substrates. The resulting concentrations of glucose, fructose, saccharose and lactic acids after fermentation of 48 h are summarized below:

Substrate 1:

Table 2 Substrate 2:

Table 3 Substrate 3:

Table 4

Strains DSM 20004 and DSM 20005 showed good yields of lactic acid in all tested substrates 1 to 3 with 9.5, 21.9, 13.8 g/L and 9.2, 23.7, 10.4 g/L lactic acid. Solely heterofermentative strain DSM 20241 showed even higher yields of 15.7 and 17.3 g/L in substrates 1 and 3.

Thus, Lactobacillus coryniformis subsp. torquens (DSM 20005) was used for producing mirabelle jam comprising D-lactic acid as shown in Example 2.

Example 2 - Method for producing mirabelle jam comprising D-lactic acid Frozen, whole mirabelles with pit were thawed completely. The mirabelles were pitted and the remaining skins and pulp were thoroughly blended to obtain a mirabelle jam. The pH of the blended Mirabelle jam was 3.2

As a preculture, Lactobacillus coryniformis subsp. torquens (DSM 20005) was inoculated into 150 mL MRS medium from three agar slant tubes and incubated 24 h at 30°C and 100 rpm. MRS medium is a liquid medium recommended for use in the cultivation of

Lactobacillus species and lactic acid bacteria.

Complex carbohydrates in the blended mirabelle fruit mass are optionally enzymatically hydrolyzed to increase the concentration of readily fermentable monosaccharides. As a substrate, 2.5 kg mirabelle jam was adjusted to 2.5 I with sterile distilled water and transferred into an empty autoclaved 5-L-fermenter. For enzymatic hydrolysis, 2.5 ml_ pectinase L-40 was added, pH was adjusted to 4.5 with 20% NaOH and hydrolysis was performed for 24 h at 50°C and a disk stirrer rotating at 500 rpm. After hydrolysis, the pH was adjusted to 6.0 with 20% NaOH.

After an incubation time of 24 h, 150 ml_ preculture containing Lactobacillus coryniformis subsp. torquens (DSM 20005) was added to the mirabelle jam into the 5-L-fermenter. Fermentation was performed for 50 h at 30°C using a blade disk stirrer rotating at 400 rpm. pH was monitored with a sensor and maintained at pH 6.0 by adding 20% NaOH through an automated pump system.

The resulting concentrations of D-lactic acid [g/L] after fermentation for 26 h and 50 h are 20.1 g/L and 40.5 g/L, respectively.

Example 3 - Determination of the bioenergetics health index 1. Introduction

Determination of the bioenergetic health index (BHI) has been suggested as a useful clinical test to monitor mitochondrial function in health and disease (Chacko et al. , Clin. Sci. 127 (2014), 367-373). Mitochondrial function is estimated by measuring the oxygen consumption rate (OCR) of cells isolated from blood samples using an extracellular flux analyzer (Seahorse Bioscience). This system allows real-time, noninvasive OCR measurement. Sequential addition of inhibitors of the mitochondrial respiratory chain allows correlation of the respective OCR to mitochondrial function and, thus, a bioenergetic profile can be determined. Based on this profile the BHI, representing a person's composite mitochondrial profile for a selected cell type, can be calculated.

Oligomycin inhibits ATP synthase (complex V) and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production. Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the ETC is uninhibited and oxygen is maximally consumed by complex IV. The FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity is a measure of the ability of the cell to respond to increased energy demand. The third injection is a mix of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of non-mitochondrial respiration driven by processes outside the mitochondria (Seahorse XF Cell Mito Stress Test Kit User Guide, Part# 103015-100, 103016-400 Rev D; Agilent Technologies).

2. Blood collection and cell preparation

Venous blood samples (1-2 tubes, 8.5 ml/tube) were collected from donors in vacutainers (BD Biosciences) containing 1.5 ml of ACD (acid citrate dextrose) solution (trisodium citrate, 22.0 g/l; citric acid, 8.0 g/l and dextrose 24.5 g/l) as anti-coagulant and processed within 2-4 h of collection. Isolation procedures were performed at room temperature and were designed to prevent cell activation. Blood was centrifuged with a swinging-bucket rotor at 500 g for 10 min at room temperature. After the platelet-rich plasma was removed, the buffy coat (leucocyte-enriched layer) on top of the red blood cell pellet was carefully collected and diluted (1:4) with basal RPMI 1640 medium (Thermo Fisher Scientific), gently layered over a Histopaque density gradient of specific gravity = 1.077/1.119 (Sigma-Aldrich) and centrifuged without brake at 700 g for 30 min. The top white ring containing the peripheral blood mononuclear cells (PBMCs) was collected, diluted 1:4 with RPMI 1640 and cells were pelleted by centrifugation at 700 g for 10 min. The PBMC pellet was resuspended in Seahorse XF DMEM assay medium (Agilent) supplemented with 5.5mM D-glucose, 4mM L-glutamine, 1mM pyruvate, and cell density was determined. Typically, isolation yielded cell populations with >85% viability as determined by Trypan Blue exclusion assay.

3. OCR Measurement

The Seahorse instrument (Agilent) is designed to work with adherent cells. According to the manufacturer's instructions and to promote adherence of PBMCs, Seahorse XF24 V7 PS Cell Culture Microplates (Agilent) were coated with 30 pi of Cell-Tak (Thermo Fisher Scientific), prepared according to manufacturer's instructions. After incubating the plates for 20-30 min at 37°C, the cell adhesive was aspirated and wells were washed twice with PBS (1 ml/well). PBMCs were plated on CellTak-coated assay plates (2.5 x 105 cells/well in 200 ml) and allowed to settle for 30-60 min at 37°C. To attach the sedimented cells to the bottom of the plate, plates were centrifuged at 40 g without brake and 660 ml XF assay medium are added. The plates were equilibrated at 37°C for 30 min in a non-C02 incubator before being transferred to the Seahorse instrument. The basal OCR was measured for 20 min before the mitochondrial stress test was performed by sequentially injecting oligomycin (2 mM), FCCP (1.5 pM), and antimycin A (5 pM) into the cellular media to achieve final concentrations of 2 pM, 1.5 pM and 5 pM, respectively. At the end of the assay period, cell lysates were collected, and OCR values normalized to the protein content in each well.

Each donor-derived PBMC sample was analyzed using 2-3 technical replicates. Data are presented as mean ± SD.

The optimal number of PBMC for OCR determination and the optimum concentration of inhibitors for the mitochondrial stress test was determined in previous pilot experiments.

4. Guantification of BHI

The OCR normalized to protein (pMoles/min/pg protein) were used to calculate ATP- linked respiration, proton leak, maximal OCR, reserve capacity and non-mitochondrial respiration. The Bioenergetic Health Index (BHI) is calculated using the following formula: BHI = (reserve capacity c ATP-linked OCR) / (proton leak c non-mitochondrial OCR).

Table 5