Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
  • G01N21/80—Indicating pH value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N2021/7769—Measurement method of reaction-produced change in sensor
  • G01N2021/7786—Fluorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
  • G01N33/04—Dairy products

Definitions

• the present invention relates to improved pH indicator systems, which are especially suitable for use in microbioreactors.
• the pH indicator systems are based on pH-sensitive fluorescent molecules.
• Acidifying bacteria are commercially utilized in many products. These bacteria are commonly provided as starter cultures to the starting material, converting the starting material to the desired product. Optimizing these cultures and introducing new cultures is of interest for the manufacturers of these products and for starter culture manufacturers. To do so, proper selection of strains with the desired growth and acidification performance is very important.
• a typical working range for acidifying bacteria is between pH 3 and 8. The proton concentration varies with a factor 100.000 in this range.
• a specific field where acidifying bacteria are used is dairy. Milk or milk products are acidified or fermented to obtain desired product properties. Moreover, acidified dairy products are better preserved. Typically milk products are acidified with lactic acid bacteria.
• cancer cells produce acid. Monitoring acid production is important for research purposes. Moreover, some yeast cells have also been known to produce different types of acid from typical carbon sources.
• Microbioreactors including microtiter plates, are used for screening of cultures. These microbioreactors provide the opportunity to screen on small scale a large variety of conditions in a highly automated fashion. Microorganisms can be cultured under different conditions whilst monitoring their growth and other parameters. Typical growth times are between 2 and 40 hours, but can be extended to weeks, so reliable growth monitoring is needed for a relative lengthy period. Typically microbial growth is followed by measuring the optical density (OD). A common method is to monitor the optical density at 600 nm (OD600).
• turbid aqueous compositions such as for example milk or milk-component comprising compositions
• optical density measurement at high cell concentrations is less reliable since optical density is less sensitive to cell concentration at these high cell concentrations. Therefore, in cases where background turbidity could disturb OD measurements and acidifying bacteria are concerned, microbial growth is preferably monitored using pH measurements with pH electrodes. Next to this, pH is preferably also measured when acidifying bacteria are concerned since often there is a need for a specific target pH for texture, taste or microbial stability reasons.
• pH electrodes are relatively large volumes. Due to these volume requirements, the number of samples that can be simultaneously analysed is limited. Moreover, pH electrodes are knowingly susceptible to drift, the membrane can be clotted (by cells or medium components), needing regular recalibration and giving potential measurement inaccuracies over the whole growth time.
• pH sensitive fluorescent probes have been developed and are being used. I n the art assemblies have been provided for pH measurements based on fluorescence. A pH sensitive fluorescent molecule or fluorophore is immobilized in a measurements assembly, sensor or probe. Protons diffuse towards the pH sensitive fluorophore in the probe.
• US 2014/0080222 describes an example wherein a fluorophore with a pH sensitive fluorescent moiety and a pH insensitive fluorescent moiety is introduced in a probe. Either light from two wavelengths is sequentially applied, and at each excitation wavelength the fluorescence is measured at a third wavelength (dual excitation/single emission) or light from one wavelength is applied and the fluorescence is measured at two wavelengths (single excitation/dual emission). General fluctuations in intensity can be compensated in this way since the ratio between the pH sensitive wavelength and the pH insensitive wavelength will not change upon intensity fluctuations.
• two luminescent materials are introduced in a sensor, one pH sensitive one not pH sensitive. Based on the shift in decay times the pH can be determined.
• a fluorophore with two pH sensitive fluorescent moieties with different pKa's is described WO 2016/071465. Fluorescence lifetime is measured.
• Microtiter plates with integrated immobilized fluorophores are commercially available (e.g. , HydroPlates HP96U, PreSens Precision Sensing GmbH, Germany). A pH measurement range between 5 and 8 is advised by the supplier.
• Other commercially available systems comprise an array of single-use vessels having a volume of 8-15 mL that can mimic the characteristics of classical lab scale bioreactors.
• the Ambr® 15 fermentation system (Sartorius Stedim) is an automated microscale bioreactor system designed for microbial screening applications. It is said to provide a consistent microscale model for early stage microbial strain screening with enhanced capabilities supporting fed-batch microbial cultures.
• the system provides an integral impeller for rapid, efficient mixing, a sparge tube for delivering gas to the impeller mixing zone, and a sample port to allow addition of reagents and feeds or removal of samples. It comes with immobilized fluorophores integrated in the microbioreactor allowing to accurately measure pH in the range of 6-8.
• the present inventors now surprisingly found that by employing a combination of different fluorophores, pH values can be reliably measured in a microbioreactor within a pH range from 3-9, preferably within a pH range from 3.5-7.5, more preferably within a pH range of 4-7. Moreover the inventors surprisingly found that during growth of micro-organisms or under the influence of chemical processes e.g. leading to acidification, in some growth media such as those comprising cow's milk, the level of auto-fluorescence of said growth media varies so as to disturb the fluorescence pH measurement, yet the inventors also surprisingly found that by introducing a pH- insensitive fluorophore emitting in the auto-fluorescence range, this disturbance can be diminished or even overcome. Also the inventors surprisingly found that by adding the fluorophores to the aqueous composition, more accurate results can be obtained. Also, the inventors surprisingly found that by a simple fluorescence intensity measurement, reliably acidification can be monitored.
• the concentration of pH sensitive and pH insensitive fluorescent compounds or moieties can easily be optimized by adapting these concentrations. So, the present inventors found that the accuracy of the method for measuring the pH of an aqueous composition, such as a growth medium, can be further improved in case the aqueous composition generates an auto-fluorescence signal that overlaps with the emission spectrum of one or more of the pH sensitive fluorescent compounds.
• the accuracy of the method can be significantly improved by adding a fluorophore that is capable of generating a fluorescence emission signal that overlaps with the emission spectrum of the auto-fluorescence signal, wherein the intensity of said fluorescence emission signal generated by the fluorophore as a function of the pH of the aqueous composition does not vary by more than 40% in a pH range spanning at least 2 pH units.
• the accuracy of the method for measuring the pH of an aqueous composition can be further improved in case the aqueous composition generates an auto-fluorescence signal at a wavelength that overlaps with a pH insensitive wavelength which is used for the fluorescence pH measurement.
• the accuracy of the method can be significantly improved by adding a fluorophore that is capable of generating a fluorescence emission signal that overlaps with the emission spectrum of the auto-fluorescence signal, wherein the intensity of said fluorescence emission signal generated by the fluorophore as a function of the pH of the aqueous composition does not vary by more than 40% in a pH range spanning at least 2 pH units.
• the auto- fluorescence signal may alter. This alteration is dependent on the specific biological process that takes place.
• a non-limiting list of variables in the biological process that may affect the auto- fluorescence are the type and concentration of cultures, the composition of the growth medium, growth conditions as temperature, concentration of oxygen and other gasses and so on. Especially the type and concentration of cultures may affect the auto-fluorescence signal.
• microorganisms and their excretions are surface active and have a tendency to block and/or invade permeable membranes and, by this mechanism, disturb proton migration and block fluorescence excitation and emittance. Also gas bubbles sticking to the probe surface can interfere with proton migration. Upon acidification gas may be formed.
• fluorescent material needs to be integrated and immobilized in a probe, specific requirements apply for such a material, limiting the choice of fluorescent moieties and the number of fluorescent molecules. Often one compound with different moieties is used as fluorescent material. Combining such moieties in one molecule is complex and expensive.
• these problems are reduced or overcome by adding the fluorophores directly to the aqueous composition. This simplifies assay design and increases accuracy since fouling issues are prevented, as there is no longer need for a probe to be in direct contact with the substance to be measured and no permeable membrane is used. Moreover it greatly enhances flexibility in choice of fluorophores and concentration of fluorophores.
• the fluorophore is integrated in a microtiter plate.
• a microtiter plate is characterized with a specific number of wells.
• the current invention frees the user of using specific microtiter plates with only a specific number of wells, offering freedom in choosing the number of wells.
• one pH-dependent fluorophore is incapable for measuring the pH over the whole range.
• the fluorescent emission of the fluorophore becomes insensitive to pH at pH levels further away from its pKa. As the emission spectrum becomes pH insensitive the pH measurement will be insensitive and therefore inaccurate.
• the inventors found that by applying two or more fluorophores, each with a different pKa, the pH can be measured accurately over a large pH-range. Even higher accuracy may be obtained if the two pH sensitive fluorophores are combined with a pH insensitive fluorophore.
• 1 nm is 1 nanometer is 1 - 10' 9 meter
• 1 pL is 1 picoliter is 1 10 "12 liter
• 1 ml_ is 1 milliliter is 1 ⁇ 10 "3 liter.
• the optical density (OD) is the negative of the natural logarithm of the quotient of the radiant flux transmitted by a material and the radiant flux received by that material.
• the OD measured at a specific wavelength is written as ODx nm, meaning the optical density at a wavelength of X nm (X nanometer).
• a turbid composition is a composition that is not completely transparent. Turbidity can be defined by an OD value; a turbid composition typically has an ⁇ exceeding 0.1 with a baseline value for the OD600 of water set at 0 (zero), typically a turbid composition typically has an ⁇ exceeding 0.2, typically a turbid composition typically has an OD600 exceeding 0.3.
• Luminescence is the emission of light by a compound or moiety not resulting from heat.
• Photoluminescence is a specific form of luminescence, for which light is emitted as a result of absorption of light or photons.
• Fluorescence is a specific form of photoluminescence caused by singlet singlet electronic relaxation. So fluorescence is a form of photoluminescence for which light is emitted as a result of absorption of light in a wavelength range capable of inducing singlet singlet electronic relaxation in the fluorophore. Fluorescence typically occurs in the range of visible and UV light. Light is absorbed by a compound or moiety, or, in other words, a compound or moiety is excited by a light source, resulting in emittance of light by the compound or moiety.
• the composition is illuminated as to excite the composition resulting in emittance of light.
• Light can be absorbed in a range of absorbance frequencies or wavelengths and light is emitted in a range of emission frequencies or wavelengths.
• the frequency or wavelength at which the maximum absorbance occurs is the maximum absorbance frequency or wavelength.
• the frequency or wavelength at which the maximum emission occurs is the maximum emission frequency or wavelength. In most cases the maximum absorption or excitation frequency is lower than the maximum emission frequency.
• Fluorescent absorbance, fluorescent emittance and fluorescent excitation are respectively absorbance, emittance and excitation related to fluorescence.
• Auto-fluorescence is the natural emission of light by biological structures when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores). Auto-fluorescence can take place at a specific range of wavelengths. Auto- fluorescence can become a problem with regard to determining the pH value, if auto-fluorescence signal intensities vary e.g. under the influence of micro-organisms.
• a marker is a compound added to a composition to facilitate the analysis of a parameter of this composition.
• a moiety or a chemical moiety is a part of a molecule or a functional group of a molecule.
• a functional group or a functionality is a group that exhibits consistently (so predominantly independent of the molecule it is attached to) a specific property.
• a fluorescent moiety is a moiety that exhibits fluorescence behaviour.
• a fluorescent moiety may by itself comprise one or more functional groups. These functional groups may be substituted (so replaced with a different functional group) without losing the functional behaviour of the fluorescent moiety. The functional behaviour of a fluorescent moiety may be altered by substitution of a functional group.
• a fluorescent compound or fluorophore is a compound that exhibits fluorescence. Such a fluorescent compound or fluorophore may comprise one or more fluorescent moieties.
• a fluorescent marker is a marker that exhibits fluorescence. Such a fluorescent marker may comprise one or more fluorescent moieties.
• Rhodamines are a family known in the art of fluorescent compounds or fluorescent moieties with a modified xanthene group as the core (Beija et al. , Chem. Soc. Rev., 2009, 38, 2410-2433). In Beija et al. (2009) the core group is represented. Compounds or moieties belonging to the rhodamine family are among others sulforhodamine B, rhodamine WT, rhodamine B, rhodamine 6G, rhodamine 19, rhodamine 101 , rhodamine 1 10, rhodamine 1 16 and rhodamine 123. When Rhodamine is referred to in this application it can be any compound or moiety of the rhodamine family unless a specific rhodamine compound or moiety is denoted.
• Fluorescein is a known in the art fluorescent compound (lUPAC name: 3', 6'- dihydroxyspiro[isobenzofuran-1 (3H),9'-[9H]xanthen]-3-one). Fluorescein can be modified as to form a moiety or its functional properties can be altered by adding or substituting functional groups (Duan et al, Minireviews in org. chem., 2009, vol 6, No. 1 , 35-43). Compound or moieties belonging to the fluorescein family are among others 5(6)-carboxyfluorescein and 2,7'-dichlorofluorescein.
• fluorescein When fluorescein is referred to in this application it can be any compound or moiety in which fluorescein is present either with or without added or substituted functional groups unless a specific fluorescein compound or moiety is denoted.
• Similar fluorescent moieties are fluorescent moieties with the same fluorescent functional group but with at least one different functional group substituted to the fluorescent functional group.
• the similar fluorescent moieties 5(6)-carboxyfluorescein and 2,7'- dichlorofluorescein comprise the same fluorescein functional group, being the same fluorescent functional group but the fluorescent functional group is substituted with different functional groups.
• a fluorescent moiety with a different substituted group may exhibit a different fluorescent behaviour.
• the absorbance spectrum and/or the emittance spectrum may alter.
• the maximum absorbance frequency or the maximum emission frequency may alter.
• a fluorescent moiety with a different substituted group may exhibit a different pKa.
• a bioreactor is a device or vessel in which a biological process takes place.
• a bioreactor may among others be used to grow cells or for biological conversions. A specific biological conversion is fermentation.
• a microbioreactor is a bioreactor with a volume typically smaller then 100 ml.
• a microbioreactor preferably has a volume in the range of 1 pL - 50 ml_, more preferably in the range of 1 nl_ - 10 mL.
• a microbioreactor may be provided with means to monitor and adapt gas concentrations, concentration of other compounds, pH, temperature and pressure.
• a microbioreactor may be provided with means to measure or monitor fluorescence.
• a microbioreactor may also be provided with means for mixing, as for example a stirrer.
• a microbioreactor may also be provided with means for taking a sample.
• Typical gasses for monitoring and adapting are air, oxygen, nitrogen and carbon dioxide, and mixtures thereof.
• Other compounds can be added in pure or dissolved form. Such a compound may be added in a mixture of compounds.
• specific substrates and growth substrates can be added.
• Acid or, preferably, base may be added.
• a suitable acid is for example hydrochloric acid.
• Suitable bases are for example ammonia, sodium hydroxide and potassium hydroxide.
• a turbid composition is a composition that is not completely transparent. Turbidity can be defined by an OD value; a turbid composition typically has an OD600 exceeding 0.1 , with a baseline value for the OD600 of water set at 0 (zero), typically a turbid composition has an OD600 exceeding 0.2, typically a turbid composition has an ⁇ exceeding 0.3. Turbid aqueous compositions may be contained in a microbioreactor.
• Turbid aqueous compositions are for example cell cultures, fermentation media, milk and blood.
• a turbid aqueous composition may be obtained by introducing a clouding agent into a transparent aqueous composition.
• a clouding agent is a compound that is capable of increasing the OD600 of a transparent aqueous composition.
• Preferred clouding agents are particles that are not soluble in the aqueous composition and form a suspension with the aqueous composition.
• the clouding agents are not fluorophores.
• the clouding agents do not alter the pH of an aqueous composition by more than 0.3 units when added to the aqueous composition which preferably has a pH in the range of 3-8.
• Preferred clouding agents are silica or titanium dioxide particles.
• the particle sizes of the silica or titanium dioxide particles are preferably chosen such that the suspension is stable over a time period during which the pH is measured.
• the turbid aqueous composition may be provided with a stabilization aid in order to prevent undue sedimentation of the clouding agent.
• a preferred stabilization aid is selected from the group consisting of a hydrocolloid and a sequestering agent; herein a preferred hydrocolloid is a polysaccharide, such as xanthan gum, guar gum or gum Arabic; a preferred sequestering agent is EDTA or NTA or their sodium salts.
• Turbid aqueous compositions herein preferably comprise blood, food slurries, milk, aqueous compositions that comprise one or more clouding agents, blood-derived components or milk- derived components.
• Turbid aqueous compositions herein most preferably comprise milk or milk- derived components.
• Milk- derived components are for example, milk protein, milk lipids, milk carbohydrates and milk salts.
• Milk proteins comprise casein and whey protein.
• Milk lipids comprise milk fats and milk phospholipids.
• Milk carbohydrates comprise lactose. Casein and fat in an aqueous composition may cause turbidity.
• the turbid aqueous compositions may comprise an added clouding agent; this is particularly preferred if the aqueous composition would comprise no milk or blood, or in case the aqueous composition have an ⁇ value of less than 0.5 or less - more preferably of 0.3 or less, most preferably of 0.1 or less - in the absence of the added clouding agent.
• Fermentation is a process in which microorganisms grow on a growth medium. Often sugars are converted into acids, gases or alcohol when fermenting.
• the growth medium may at the start of fermentation be turbid or non-turbid.
• the invention thus concerns a method for measuring the pH of an aqueous composition comprising: providing an aqueous composition comprising
• a pH insensitive fluorophore which does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 20% in a pH range spanning at least 2 pH units,
• aqueous composition is preferably a turbid aqueous composition.
• the invention concerns a method for measuring the pH of an aqueous composition comprising: providing an aqueous composition comprising
• a pH insensitive fluorophore which does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 20% in a pH range spanning at least 2 pH units,
• aqueous composition is a turbid aqueous composition.
• the invention concerns an aqueous fluorescent pH indicator composition
• aqueous fluorescent pH indicator composition comprising a first pH sensitive fluorophore and a pH insensitive fluorophore which does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 20% in a pH range spanning at least 2 pH units.
• the aqueous composition may contain an added clouding agent, such as silica particles or titanium dioxide particles.
• the aqueous composition comprises an added clouding agent, preferably the added clouding agent is one of silica particles or titanium dioxide particles.
• the concentration of the added clouding agent in the aqueous fluorescent pH indicator composition is such to increase the OD600 value of the aqueous composition by at least 0.1 units, more preferably by at least 0.3 units, most preferably by at least 0.5 units.
• kits-of-parts comprising a microbioreactor and an aqueous fluorescent pH indicator composition comprising a first pH sensitive fluorophore and a pH insensitive fluorophore which does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 20% in a pH range spanning at least 2 pH units.
• the aqueous fluorescent pH indicator composition comprises a second pH sensitive fluorophore.
• the pKa value of the first pH sensitive fluorophore is in the range of 4 to 6, and the pKa value of the second pH sensitive fluorophore is in the range of 5 to 7.
• the first pH sensitive fluorophore and the pH insensitive fluorophore are excitable at the same wavelength.
• the aqueous composition may contain an added clouding agent, such as silica particles or titanium dioxide particles.
• the aqueous composition comprises an added clouding agent, preferably the added clouding agent is one of silica particles or titanium dioxide particles.
• the concentration of the added clouding agent in the aqueous fluorescent pH indicator composition is such to increase the OD600 value of the aqueous composition by at least 0.1 units, more preferably by at least 0.3 units, most preferably by at least 0.5 units.
• the pH of the composition can be determined by comparing the measured fluorescence emission intensity with the fluorescence emission intensity of a calibration curve.
• the calibration curve typically correlates fluorescence to pH.
• the measured fluorescence intensity may be measured at one or at more wavelengths.
• the calibration curve may be created over a pH range that is relevant for the pH range to be measured.
• a calibration curve can be made relating pH to fluorescence by measuring fluorescence at different pH's. Typically this measurement is done at a wavelength at which the pH- sensitive fluorophore has a strong signal and at which the signal changes upon a pH change; the pH-sensitive wavelength. Improved accuracy can be obtained by also measuring fluorescence at a pH-insensitive wavelength in addition to the pH-sensitive wavelength. By taking the ratio of the measurement at these wavelengths, background fluctuations can be extinguished.
• a calibration curve can be made for a calibration based on a measurement at a pH-sensitive wavelength and a measurement at a pH-insensitive wavelength (the pH sensitive wavelength) by relating pH to the fluorescence ratio at different pH's.
• Fluorescence can be measured as fluorescence intensity (I).
• the reference pH can be measured by a conventional pH measurement as for example a pH electrode.
• the calibration curve is made in an aqueous composition that is similar to the composition to be measured.
• An aqueous composition that is similar comprises the same components as the aqueous composition to be measured or at least comprises the components of the aqueous composition which are relevant for the fluorescence measurement for the purpose of pH determination and the concentration of these components are in a similar range of the aqueous composition to be measured.
• the concentration of these components is within 30% (w/w) of the concentration of the components in the aqueous composition to be measured, more preferably within 20% (w/w) and even more preferably within 10% (w/w) of the components in the aqueous composition to be measured.
• Components which are relevant for the fluorescence measurement comprise salts and compounds comprising fluorescent moieties.
• fluorescence can be measured through a single excitation/dual emission measurement and/or a dual excitation/single emission measurement.
• the method for measuring the pH of an aqueous composition optionally comprises providing a second pH sensitive fluorophore.
• the pKa of the second pH sensitive fluorophore is within the intended pH measurement range.
• the aqueous composition optionally preferably comprises a second pH sensitive fluorophore, wherein the first pH sensitive fluorophore and the second pH sensitive fluorophore comprise different fluorescent moieties and/or comprise similar fluorescent moieties comprising different functional groups.
• the first pH sensitive fluorophore and the second pH sensitive fluorophore each have different pKa values, preferably wherein the first pH sensitive fluorophore has a first pKa value and the second pH sensitive fluorophore has a second pKa value, and wherein the first pKa value differs from the second pKa value by at least 0.5 units, more preferably by at least 1 unit.
• the first pKa value differs from the second pKa value by at least 1.5 units, most preferably by at least 2 units.
• the fluorescent moieties are selected from the group consisting of fluorescein moieties and rhodamine moieties.
• the first pH sensitive fluorophore, the second pH sensitive fluorophore and/or the pH insensitive fluorophore each comprise one or more fluorescent moieties.
• the one or more fluorescent moieties are selected from the group consisting of fluorescein moieties and rhodamine moieties.
• the pKa value of the first pH sensitive fluorophore is in the range of 4 to 6, and the pKa value of the second pH sensitive fluorophore is in the range of 5 to 7.
• the first pH sensitive fluorophore and the pH insensitive fluorophore are excitable at the same wavelength.
• the aqueous composition comprises milk components, preferably casein.
• the aqueous composition exhibits auto-fluorescence in the emission range between 600-620 nm.
• the method for measuring the pH of an aqueous composition comprises providing a pH insensitive fluorophore which does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 20% in a pH range spanning at least 2 pH units.
• a pH insensitive fluorophore does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 30%, preferably by more than 20% in a pH range spanning at least 2 pH units, preferably at least 3 pH units.
• a pH sensitive fluorophore is a fluorophore that is not pH insensitive.
• the terms pH sensitive and pH insensitive relate to pH sensitivity in the relevant pH measurement range, being the pH range of interest or the intended pH measurement range.
• the use of a pH insensitive fluorophore is especially useful if the aqueous composition has a certain autofluorescence signal which overlaps with the emission spectrum at the pH-insensitive wavelength.
• the use of a pH insensitive fluorophore is particularly useful if the aqueous composition has a certain autofluorescence signal that varies due to a process that takes place in the aqueous composition, more specifically due to a biological process that takes place in the aqueous composition, even more specifically due to a microbial process that takes place in the aqueous composition.
• a pH insensitive fluorophore is exceptionally useful if the aqueous composition has a certain autofluorescence signal that varies due to a process that takes place in the aqueous composition and has a certain autofluorescence signal which overlaps with the emission spectrum at the pH-insensitive wavelength.
• the pH insensitive fluorophore is capable of generating a fluorescence emission signal that overlaps with the emission wavelength range of the auto-fluorescence signal of the composition.
• the pH insensitive fluorophore has a maximum emission wavelength which differs at least 10 nm, more preferably at least 20 nm, most preferably at least 30 nm from the maximum emission wavelength of the first pH sensitive fluorophore.
• the pH insensitive fluorophore has a maximum emission wavelength which differs at least 10 nm, more preferably at least 20 nm, most preferably at least 30 nm from the maximum emission wavelength of the second pH sensitive fluorophore.
• the concentration of the pH insensitive fluorophore should be such that the emission intensity at a wavelength relevant for the pH insensitive fluorophore exceeds the autofluorescence intensity by at least two times the emission intensity of the aqueous composition without additives at that wavelength.
• the wavelength relevant for the pH insensitive fluorophore is the wavelength at which the emission intensity is measured of the pH insensitive fluorophore.
• the additives are the fluorophores or other compound that provide a fluorescence signal.
• the concentration of the pH insensitive fluorophore in the aqueous composition is such that the emission intensity at a wavelength relevant for the pH insensitive fluorophore exceeds the autofluorescence intensity by at least two times the emission intensity of the aqueous composition without additives at that wavelength, more preferably by at least three times, even more preferably by at least four times.
• the skilled person can adapt the concentration of the pH insensitive fluorophore to exceed the autofluorescence emission intensity of the aqueous composition by a certain level.
• the skilled person may alternatively first measure the autofluoresence emission spectrum of the aqueous composition and based on this measurement then decides which pH insensitive fluorophore to use, wherein the choice relates to the autofluorescence emission spectrum.
• the aqueous composition is turbid, or in other words preferably the aqueous composition is a turbid aqueous composition.
• Turbid aqueous compositions herein preferably comprise blood, food slurries, milk, one or more clouding agents, blood-derived components or milk-derived components, more preferably one or more clouding agents, milk or milk-derived components, most preferably milk or milk-derived components.
• the aqueous composition comprises blood, food slurries, milk, one or more clouding agents, blood-derived components or milk-derived components, more preferably one or more clouding agents, milk or milk-derived components, most preferably milk or milk-derived components.
• the turbid aqueous composition preferably further comprises a microorganism, most preferably a cancer cell or a bacterium that is capable of producing an acid when grown on a suitable carbon source.
• the acid preferably comprises lactic acid and the suitable carbon source preferably comprises glucose or lactose, most preferably lactose.
• the cell count density of the microorganism in the turbid aqueous composition is preferably at least 100 cells/mL, more preferably at least 1000 cells/ml, even more preferably 10,000 cells/ml, most preferably at least 100,000 cells/mL.
• the expression "cells/mL” may be read as "cfu/mL" in case of bacteria.
• the bacterium is preferably selected from the group consisting of Lactococcus, Lactobacilus, Streptococcus thermophilus, Oenococcus and Leuconostoc.
• the measured fluorescence intensity may be affected by the sample that is measured.
• the light of the excitation source may be scattered or absorbed by other compounds than the fluorophore that are present in the sample or the emitted light by the fluorophore may be scattered or absorbed by other compounds that are present in the sample.
• the energy that is absorbed by a fluorophore may be transferred to other compounds in the aqueous composition (quenching).
• incoming or excitation light may be absorbed or scattered. This effect is different from autofluorescence; for autofluorescence other compounds than the added fluorophores demonstrate fluorescence.
• Quenching is also different from autofluorescence as it concerns the transferal of the energy that is absorbed by a fluorophore to other compounds in the aqueous composition.
• any reference made to quenching and/or turbidity in relation to measurement of pH with a fluorescent indicator does not imply autofluorescence and does not imply the recognition of problems related to autofluorescence in relation to measurement of pH with a fluorescent indicator.
• an aqueous composition of which the pH is monitored by using fluorescence measurements may change in turbidity.
• an aqueous composition with initially a low turbidity or no turbidity may demonstrate increased turbidity because of cell growth. It is common to use culture medium with low or no turbidity to grow cells.
• the increasing turbidity may affect the measurement by absorbing or scattering part of the light coming from the excitation source or absorbing or scattering part of the light emitted by the fluorophore, resulting in a less accurate pH measurement.
• a clouding agent may be added to increase the initial turbidity. I n this way a constant high turbidity is obtained which improves the accuracy of the pH measurement.
• the aqueous composition is milk and carboxyfluorescein and/or dichlorofluorescein are used as pH sensitive fluorophores at an excitation wavelength of 495 nm; the ratio of the emission intensity is preferably measured at 535 and 610 nm.
• the 1535: 1610 ratio varies between 2 and 18 in the pH range of 3.8-7.0 and by generating a calibration curve correlating pH and emission intensity ratio, the 1535: 1610 ratio can be used to predict the pH in this turbid aqueous composition.
• Milk typically shows auto-fluorescence (emission) near 610 nm. During fermentation this signal can be reduced so that the measured 535/610 ratio becomes a less accurate predictor of pH.
• the present inventors found that in such cases if the predicted pH value differs from the actual pH value (measured using a pH electrode) by more than 0.2 pH units in a range of pH 3.8-5.5 or in a range of pH 5.5-7 or in a range of pH 3.8-7, the accuracy of the pH prediction can be improved by adding a pH insensitive fluorophore.
• the predicted pH value differs from the actual pH value (measured using a pH electrode) by more than 0.05 or more than 0.1 pH units in a range of pH 3.8-5.5 or in a range of pH 5.5-7 or in a range of pH 3.8-7, the accuracy of the pH prediction can be improved by adding a pH insensitive fluorophore.
• Rhodamine is characterized by a 1535:1610 ratio which varies by not more than 0.2 (preferably between 0.9 and 1 .1 ) within the pH range of 3.8-7.0.
• Rhodamine is preferably added to a milk-based aqueous composition in a concentration such that the emission intensity of the resulting mixture when measured at 610 nm is increased by a factor of at least two as compared to the same aqueous composition without the rhodamine or added pH sensitive fluorophores.
• the invention concerns a method for measuring the pH of an aqueous composition
• a method for measuring the pH of an aqueous composition comprising a) providing an aqueous composition comprising i) a first pH sensitive fluorophore and ii) a pH insensitive fluorophore which does not vary in intensity of the emitted fluorescence signal by more than 40%, preferably by more than 20% in a pH range spanning at least 2 pH units, b) illuminating the aqueous composition so as to excite the composition, c) detecting light emitted by the composition, and d) determining the pH of the composition based on the fluorescent emission intensity, wherein the aqueous composition is preferably turbid, or preferably a turbid aqueous composition and/or wherein the aqueous composition preferably comprises milk or blood, preferably milk.
• pH sensitive fluorophores Preferred characteristics of pH sensitive fluorophores are as following.
• IR emission ratio (measured at two different wavelengths) which when measured at pH1 differs from the same ratio measured at pH2 by more than a factor 2 wherein pH1 differs from pH2 by preferably at least one pH unit.
• pH insensitive fluorophores are as following.
• pH3 is preferably within the same range (+/- 0.3 pH units) as pH1 and pH4 is preferably within the same range (+/- 0.3 pH units) as pH2, pH 1 and pH2 being the lower and the higher end of the pH range which can be predicted by the pH sensitive fluorophores as outlined above.
• the pH insensitive fluorophore can enhance a fluorescence emission signal generated by an aqueous composition (such as a growth medium, in particular a milk-based growth medium) per se by a factor of at least 2, wherein the fluorescence emission signal generated by the aqueous composition is relevant for relevant for predicting the pH by a pH sensitive fluorophore.
• an aqueous composition such as a growth medium, in particular a milk-based growth medium
• a 1 mM solution of 5(6)-carboxyfluorescein (Sigma Aldrich; #21877) and 2,7'-dichlorofluorescein (sigma Aldrich; D6665) was made by dissolving 38 mg and 44 mg, respectively, in 100 ml Tris HCI (pH 8.0; 100 mM).
• a 2 mM solution of Rhodamine B (Sigma Aldrich; #83689) was made by dissolving 82 mg in 100 ml water (demineralized).
• S-milk (now referred to as milk) was purchased from Tritium Microbiology (Eindhoven, the Netherlands).
• D-(+)-Gluconic acid ⁇ -lactone (GDL) was used to make the milk standards with the desired pH.
• GDL Sigma Aldrich, G4750
• GDL was used at a concentration of 0.1 , 0.2, 0.4, 0.5, 0.7, 0.9 1.1 , 1.3, 1 .6 and 2.0% for the 8 types of milk, one tube was used without GDL and to one tube 1.4% 1 M HCL was added.
• the glucono delta lactone was weight in a 50 ml tube and 25 ml of milk was added to the tubes.
• 1 % chloramphenicol (10mg/L) was added to the standards.
• Chloramphenicol (Sigma Aldrich, C0378) was dissolved in 50% ethanol and filter sterilized over a 0.22 pm filter. Chloramphenicol was shown to have no effect on the fluorescent signal.
• the tubes were mixed well and were placed overnight at 20°C in a stove. The day after the standards were mixed thoroughly and transferred to a 96-wells deepwell plate.
• the content of the deepwell plate was transferred to a transparent 96-well plates (200 ⁇ ) and the two empty quadrants of the black 384-well plates (80 ⁇ ).
• the standards were determined for the 96 and 384 well plate using a microtiterreader (Tecan; Infinite Pro 200) at 30°C.
• Table 2 List of used combination of fluorophores and used concentration in S-milk
• a part of the content of the deepwell plate was correspondingly transferred (using a 96 automated pipetting unit (CyBio, Selma, Analitik Jena)) to a transparent 96- well microtiterplate (200 ⁇ ; Greiner Bio-one #655 180) and to 2 quadrants of a black 384-well plate (80 ⁇ Porvair #324022). Note that in the 384-well plate there are still 2 quadrants available for the standards (these will be added the day after). All plates were sealed with an aluminum seal to prevent evaporation and to prevent light to break the fluorophores. Growth was allowed overnight at 30°C in a stove (96-well plate) and at 30°C the 384-well plate in a microtiterreader (Tecan; Infinite Pro 200). All fermentations were performed on two consecutive days in duplicate. Analysis
• the final pH was measured by a microelectrode (Metrohm, 6.0224.100) in each well of the two 96 well plates (fermented and standards). The pH measurement was finished within 20 minutes after the fluorescence of the plate was determined. The fluorescence was determined in microtiterreader (Tecan; Infinite Pro 200) with and excitation wavelength of 494nm and an emissions measurement at 535 and 610nm. A relative emission (IR ratio) was used by dividing the fluorescence at 535nm by the fluorescence at 610nm. The data was analysed using statistical software R (http://www.r- project.org/).
• Hydroplate HP96U (PreSens) were used. Hydroplates were used according to the user manual, however, to make the method better suitable for milk fermentation we have used: i) milk buffers (modified using GDL; see paragraph 'preparation of milk standards') instead of clear buffers for calibration and ii) we have narrowed the pH range from pH 4.0 to 7.0. Calibration took place according the above described method. For all measurements the hydroplates were filled without fluorophores ("no fluorophores").
• thermophilus strains (CSK0058 and CSK0069) when compared to the other codes (containing L. lactis). We could further improve the prediction further by combining "dichlo” and “carb+dichlo” with “rho". In the absence of "rho” it was observed that CSK0065, CSK0076 and CSK0120 "consume” a bit of the fluorescent signal at 610 nm (results not shown), whereas the S. thermophilus strains lack this capability. We can mask this "consumption" by CSK0065, CSK0076 and CSK0120 by addition of rhodamine B.
• Figure 1 shows the pH development (acidification curve) in time for CSK0058 in the presence of catalase with different fluorophores
• Figure 2 shows the pH development (acidification curve) in time for CSK0076 in the presence of catalase with different fluorophores A) carb, B) carb + rho, C) dichlo, D) dichlo + rho, E) carb + dichlo, F) carb + dichlo + rho.
• dichlorofluorescein and Rhodamine B results in a method that allows good monitoring of acidification curves in milk and excellent prediction of final pH values. Monitoring of pH over a larger range can be improved by adding a second pH sensitive fluorophore 5,6 carboxyfluoresceine. This method is superior when compared to HydroPlates.
• a standard growth medium for lactic acid bacteria was prepared (GM17).
• the growth medium was incubated with precultured L. lactis strain MG1363 supplied by CSK (1 %) and carboxyfluorescein (10 ⁇ ) was added.
• the OD600 of the incubated growth medium was measured and found to be lower than 0.1.
• 0.5 litre of the incubated growth medium was fermented at 30°C in a fermenter and the pH was monitored, both through conventional pH electrode measurement as through fluorescence measurement (Ex 485 nm, Em 520-535 nm). For the fluorescence measurement samples were taken and measured.
• a calibration curve relating the fluorescence data to pH was made by measuring the fluorescence of carboxyfluorescein in GM17 at different pH's.
• Fluorescence of the fermentate was measured, but also fluorescence was measured of the supernatant of the fermentate after centrifugation.
• a comparison of the conventional pH measurement with the fluorescence measurements demonstrated that the fluorescent pH measurement of the supernatant was within 0. 1 pH unit of the conventional pH measurement, whilst the fluorescent pH measurement of the fermentate started deviating from the conventional after 3 hours of fermentation; after 4 hours the pH was and remained 0.3 pH units lower than the conventional pH measurement.
• the OD rose from 0.8 at 3 hours to 3.2 at the end of fermentation.
• GM17 (clear medium) was replaced by milk, the turbidity increase as a consequence of microbial growth affected the fluorimetric pH measurement far less. It was also found that the turbidity interference could alternatively by reduced by adding a clouding agent to the GM17, such as a suspension of inert and insoluble particles capable of scattering light having a wavelength of 600 nm.
• a clouding agent such as a suspension of inert and insoluble particles capable of scattering light having a wavelength of 600 nm.

Landscapes

• Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Plasma & Fusion (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=62555008

Family Applications (1)

Country Status (2)

Family Cites Families (5)

• 2018
  • 2018-04-25 WO PCT/EP2018/000219 patent/WO2018197042A1/en unknown
  • 2018-04-25 EP EP18729560.5A patent/EP3615923A1/de not_active Withdrawn

Also Published As

Similar Documents

Legal Events