Method for enhancing the ability of a bacterial cell to use lactose

Abstract

The present invention relates to an in vitro method for enhancing the ability of urease-positive or urease-negative bacterial cells to more efficiently consume the lactose present in the environment in which they are located. The present invention further relates to bacterial cells obtained with this method, a food product and a food supplement comprising them, as well as the use thereof.

Description

[0001] “Method for enhancing the ability of a bacterial cell to use lactose” ★★★★★★★

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to an in vitro method for enhancing the ability of bacterial cells to more efficiently consume the lactose present in the environment in which they are located, in particular a urease-positive or urease-negative bacterial cell species, and bacterial cells obtained with that method.

[0005] Furthermore, the present invention relates to a food product and a food supplement which comprise said bacterial cells.

[0006] STATE OF THE ART

[0007] Lactose intolerance is a condition affecting about 60% of the population and consists in an inability of the body to completely digest the sugar present in milk and its derivatives. Lactose intolerance manifests itself with gastrointestinal disorders such as, for example, abdominal bloating, cramp-like pain, diarrhoea and, in some cases, nausea.

[0008] There exist four types of lactose intolerance: primary (the most common), secondary, developmental and congenital.

[0009] Primary lactose intolerance occurs when the amount of lactase decreases with aging. In this regard, it is estimated that the majority of adults (about 65-70% of the world population) is lactose intolerant.

[0010] Secondary lactose intolerance, less common, is due to small intestine injuries caused, for example, by inflammatory intestinal diseases, Coeliac disease, or infections.

[0011] Developmental lactose intolerance manifests itself during development may occur in premature infants.

[0012] Finally, congenital lactose intolerance is an extremely rare genetic disease that manifests itself from birth and is due to low or lack of production of the enzyme lactase. To date, lactose intolerance has mainly been treated by a change in diet, where affected individuals must substantially eliminate all sources of lactose from their diet. As a consequence, lactose intolerant individuals, who must necessarily eliminate food products containing lactose from their diet, lose the nutritional benefits brought by the consumption of foods comprising lactose.

[0013] One of the alternatives to eliminating lactose from the diet is to take supplements based on the enzyme lactase to favour the digestion of lactose.

[0014] Furthermore, in the market today there are various types of lactose-free milk and various types of plant-based milk (produced from white rice, almonds or soy) which, however, have a flavour that is distinctly different from that of classic fresh milk, as well as nutritional values that are decidedly reduced compared to milk containing lactose.

[0015] It is necessary, therefore, to provide a product with high lactose digestibility that can be used by individuals who have lactose intolerance, as well as by individuals with stomach and intestinal problems, for example individuals having an infection caused by Helicobacter pylori or affected by irritable bowel syndrome.

[0016] SUMMARY OF THE INVENTION

[0017] A first aspect of the present invention relates to an in vitro method for enhancing the ability of urease-positive or urease-negative bacterial cells to more efficiently consume lactose present in a solution, said method comprising at least one step of maintaining the urease-positive or ureasenegative bacterial cells in culture in a solution comprising urea and / or NH3 in the absence of a carbon source, thereby obtaining bacterial cells with an enhanced ability to consume the lactose present in the solution. Preferably, the method does not envisage the addition or use of antibiotics.

[0018] A second aspect of the present invention relates to bacterial cells with an enhanced ability to more efficiently consume the lactose present in the environment in which they are located, obtained with the in vitro method according to the first aspect of the invention.

[0019] A third aspect of the present invention relates to a food product comprising bacterial cells as described above, preferably selected from milk and its derivatives, such as cheese, yogurt and ice cream.

[0020] A fourth aspect of the present invention is a food supplement comprising bacterial cells characterised by an enhanced lactose-consuming efficiency, as defined above.

[0021] The food supplement can be advantageously used for the prevention of lactose intolerance in one or more individuals who suffer from it.

[0022] A fifth aspect of the present invention relates to a method for reducing the lactose concentration in a product containing it. Preferably, said method comprises at least one step of adding the bacterial cells described above in detail to the product. Preferably, said product is a food product, more preferably selected from milk and its derivatives such as, for example, a cheese, yogurt or ice cream.

[0023] DEFINITIONS

[0024] In the context of the present invention, the expression “bacterial cells with an enhanced ability to more efficiently consume the lactose present in the environment in which they are located” is meant to indicate bacterial cells subjected to the in vitro method according to the invention and which, as a consequence thereof, are capable of internalising greater amounts of lactose than those internalised and converted into lactic acid by cells not treated with the in vitro method of the present patent application.

[0025] As will specified below, said cells show a higher activation of phosphofructokinase (PFK), a glycolysis enzyme belonging to the transferase class, which leads metabolism towards the production of lactic acid. In the experimental part and in the appended figures, the bacterial cells with an enhanced ability to convert lactose into lactic acid can be indicated by the acronym “MaCs” (metabolically activated cells).

[0026] According to the invention, the bacterial cells with an enhanced ability to convert lactose into lactic acid are urease-positive or urease-negative bacterial cells.

[0027] In the context of the present invention, the expression “urease-positive bacterial cells” is meant to indicate bacterial cells that express the enzyme urease. Whereas the expression “urease-negative bacterial cells” is meant to indicate bacterial cells that do not express the enzyme urease.

[0028] In one embodiment, said bacterial cells belong to the genus Streptococcus. More preferably, the bacterial cell subjected to the in vitro method disclosed herein is Streptococcus thermophilus.

[0029] In the present invention, Streptococcus thermophilus can also be indicated as “S. thermophilus”.

[0030] In the experimental part and in the appended figures, the cells of S. thermophilus having an enhanced lactose-converting ability can be indicated with the acronym “MaCs” (metabolically activated cells).

[0031] DESCRIPTION OF THE FIGURES

[0032] Figure 1 shows the results of the experiment described in example 3. In particular:

[0033] - MaCs-Lact indicates MaCs of S. thermophilus exposed to lactose.

[0034] - MaCs-Lact-U indicates MaCs of S. thermophilus exposed to lactose and urea.

[0035] The results are shown as the mean of two replicates. The error bars represent the standard deviation.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] A first aspect of the present invention relates to an in vitro method for enhancing the ability of urease-positive bacterial cells to consume the lactose present in a solution; said method comprises at least one step of maintaining urease-positive bacterial cells in culture in a solution comprising urea and / or NH3 in the absence of a carbon source, thereby obtaining bacterial cells with an enhanced ability to convert lactose into lactic acid.

[0038] In a preferred embodiment, said method comprises, before the step of maintaining in culture in a solution comprising urea and / or NH3, at least one step of maintaining urease-positive bacterial cells in culture in the presence of at least one carbon source, preferably in the presence of lactose.

[0039] The steps of maintaining the bacterial cells in culture are implemented with techniques known to the person skilled in the art.

[0040] The urease-positive bacterial cells express the enzyme urease.

[0041] In particular, urease is an enzyme belonging to the hydrolase class which catalyses the hydrolysis of urea into carbon dioxide and ammonia with the following reaction:

[0042] (NH2)2CO + H2O = CO2 + 2 NH3

[0043] In one embodiment, said bacterial cells belong to at least one genus selected from: Streptococcus, Fructilactobacillus, Lactococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus, Pediococcus and combinations thereof.

[0044] In a preferred embodiment, the bacterial cells belong to the genus Streptococcus.

[0045] In a preferred embodiment, said urease-positive bacterial cells belong to the species Streptococcus thermophilus.

[0046] Streptococcus thermophilus is an aerotolerant anaerobic Gram-positive species that can be found in milk and in many natural cultures for producing cheese and yogurt.

[0047] As shown in example 2, the cells of S.thermophilus subjected to the method according to the present invention are capable of converting a larger amount of lactose into lactic acid (see “lyophilised MIM078 MaCs 1 billion / ml” vs “lyophilised MIM078 non-MaCs 1 billion / ml” in Table 1 ).

[0048] Preferably, the solution comprises a urea concentration between 0.5 mM and 5 mM, more preferably between 1 .5 mM and 4 mM.

[0049] In a more preferred embodiment, the solution comprises a urea concentration between 2 mM and 3 mM.

[0050] As an alternative, or in addition to urea, the solution comprises NH3.

[0051] Preferably, the solution comprises a concentration of NH3 between 1 mM and 10 mM, more preferably between 3 mM and 8 mM.

[0052] Even more preferably, the concentration of NH3 in said solution is between 4 mM and 6 mM.

[0053] Once in the cells, 1 mol of urea is hydrolysed by the enzyme urease, generating 2 moles of NH3 and 1 mole of CO2.

[0054] Without wishing to be bound by any theory, the Applicant hypothesises that the NH3 generated by the hydrolysis of urea or which spreads inside the cell goes into equilibrium with its protonated form NH4+, thus alkalinising the cytoplasm and stimulating glycolysis.

[0055] When the stimulation of glycolysis occurs in the absence of lactose or other carbon sources, the cells are depleted of glycolytic intermediaries and lactose is converted into lactic acid.

[0056] In a preferred embodiment of the invention, the method does not envisage the use and / or addition of antibiotics. In other words, the method of the present invention does not envisage the use of any antibiotic, in particular chloramphenicol and / or rifamycin.

[0057] The Applicant hypothesises that NH3 and NH4+are positive effectors of phosphofructokinase.

[0058] Therefore, lactose is converted into lactic acid, and this causes a continual demand for lactose, which is internalised and converted into lactic acid.

[0059] It follows that the cells are able to internalise the lactose and convert it into lactic acid more rapidly.

[0060] In the method according to the invention, the step of maintaining bacterial cells in culture in a solution comprising urea and / or NH3 in the absence of a carbon source, preferably in the absence of lactose, is carried out for a time between 15 and 40 minutes, preferably between 20 and 35 minutes.

[0061] In an even more preferred embodiment, said step of maintaining in culture is carried out for a time between 25 and 30 minutes.

[0062] After the step of maintaining in culture, the method preferably comprises a step of removing excess molecules of NH3 until reaching a pH between 6.5 and 8.5.

[0063] The bacterial cells obtained according to the in vitro method described thus far have shown a greater ability to convert lactose into lactic acid compared to cells not subjected to that method.

[0064] Therefore, a second aspect of the present invention relates to bacterial cells with an enhanced ability to convert lactose into lactic acid, obtained with the method described above in detail.

[0065] In particular, these cells have the enzyme phosphofructokinase (PFK) in its highest state of activation.

[0066] In particular, PFK is activated by urea and / or NH3 and, in its highest state of activation, is capable of leading metabolism towards the production of lactic acid: lactose is converted into lactic acid and this causes a continual demand for lactose by the cell and the lactose continues to be internalised and converted into lactic acid more rapidly.

[0067] The Applicant has advantageously observed that the bacterial cells subjected to the in vitro method according to the invention in a cell density greater than 1 *108cfu / ml, preferably greater than 1 *109cfu / ml in a composition comprising lactose, are adapted to reduce the lactose concentration in the composition by between 15 and 35% weight / weight.

[0068] Preferably, said cells, in the above-mentioned cell densities, are adapted to reduce the lactose concentration in the composition by between 20 and 30% weight / weight in a time between 15 and 60 min at 37 °C (see Table 1 and example 2).

[0069] In one embodiment, said bacterial cells belong to at least one genus selected from: Streptococcus, Fructilactobacillus, Lactococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus, Pediococcus and combinations thereof.

[0070] In a preferred embodiment, the bacterial cells belong to the genus Streptococcus.

[0071] In a preferred embodiment, said urease-positive bacterial cells belong to the species Streptococcus thermophilus.

[0072] Advantageously, the bacterial cells with an enhanced lactose-converting ability or a composition comprising them can be used as a medicament. The Applicant has hypothesised that, during transit from the oral cavity to the gastrointestinal tract, the bacterial cells according to the invention may receive a further metabolic activation, in view of the fact that urea is secreted in saliva and in the stomach.

[0073] Advantageously, the bacterial cells with an enhanced ability to convert lactose into lactic acid obtained with the in vitro method described above in detail can be used to produce foods comprising milk or its derivatives, such as yogurt and cheese, characterised by low amounts of lactose and thus also digestible by lactose-intolerant persons.

[0074] Therefore, a third aspect of the present invention relates to a food product comprising bacterial cells as described above.

[0075] In particular, the food product comprises cells capable of converting lactose into lactic acid more rapidly and in larger amounts.

[0076] Preferably, the food product can be selected from milk and its derivatives such as, for example, cheese, yogurt, or ice cream.

[0077] By way of example, the cheese can be Asiago, provolone, Emmental, Gruyere, Montasio, mozzarella, stracchino, or taleggio.

[0078] More preferably, said food product is yogurt.

[0079] In one embodiment, said food product can comprise additional microorganisms, preferably additional bacterial cells, even more preferably probiotic bacteria. Probiotics are live microorganisms which, administered in a suitable amount, provide a benefit to the host’s health. For example, lactobacilli and bifidobacteria are the most common types of probiotic microorganisms.

[0080] A fourth aspect of the present invention is a food supplement comprising the bacterial cells characterised by an enhanced lactose-consuming ability as defined above.

[0081] In one embodiment, the food supplement according to the invention comprises bacterial cells characterised by a high lactose-consuming ability in an amount of between 5 and 20 g / l, preferably between 10 and 15 g / l. This food supplement can be advantageously used to prevent lactose intolerance in one or more individuals who suffer from it.

[0082] A fifth aspect of the present invention relates to a method for reducing the lactose concentration in a product comprising it. Preferably, said method comprises at least one step of adding bacterial cells as described above in detail to the product. Preferably, said product is a food product. More preferably, the food product can be selected from milk and its derivatives such as, for example, a cheese, yogurt or ice cream.

[0083] More preferably, said food product is a yogurt.

[0084] In one embodiment, the product, preferably the food product, has bacterial cells added at a density between 1 *106cfu / ml and 1 *108cfu / ml, preferably greater than 1 *107cfu / ml.

[0085] In one embodiment, said product, after having had bacterial cells added to it, is maintained at a temperature between 35 and 45°C, preferably between 38 and 42°C, for a time between 20 and 90 minutes.

[0086] EXAMPLES

[0087] The present invention will now be illustrated with non-limiting, merely illustrative examples of one of the embodiments of the present invention and taking into consideration cells of S. thermophilus.

[0088] In particular, in the examples that follow, the cells of S. thermophilus subjected to the in vitro method, and thus having an enhanced ability to convert lactose into lactic acid, are indicated by the acronym “MaCs”.

[0089] Example 1 - Production of cells of S. thermophilus with an enhanced ability to consume lactose and produce lactic acid (MaCs)

[0090] For the production of cells having an enhanced ability to consume lactose and produce lactic acid, 1 *107cfu / ml of S. thermophilus MIM078 were cultured at 37 °C in an M17 culture medium (20 g / litre, lactose, 1 I), collected in the late phase of exponential growth (OD600=2.5; pH 5.8) and subjected to centrifugation (6000 rpm, 10 m).

[0091] The cells were then washed with a saline solution (9 g / l NaCI), collected by centrifugation (6000 rpm, 10 m), and suspended in 100 ml or 1000 ml of saline solution supplemented with 3 mM urea or 6 mM NH3.

[0092] After 30 minutes of incubation, the cell suspension was washed several times with a saline solution to eliminate the excess ammonia until reaching a pH between 6.5 and 8.5. The cells were concentrated to a final density between 1 *109cfu / ml and 1 *101° cfu / ml.

[0093] At this point, sucrose was added at a final concentration of 6% (w / v), frozen at -80 °C and lyophilised.

[0094] Example 2 - Reduction of the percentage of lactose in milk by MaCs of S. thermophilus

[0095] In order to evaluate the lactose-converting ability of cells obtained as described in example 1 , the MaCs of S. thermophilus MIM078 were tested in reconstituted skim milk (Difco), with a different final cell density, at 37 °C for 30 min.

[0096] In particular, both MaCs not subjected to lyophilisation and MaCs subjected to lyophilisation were tested (Table 1 ).

[0097] Table 1 : Lactose reduction by MaCs of S. thermophilus, Strain:

[0098] MIM078

[0099] *Lactose reduction is calculated using the lactose content in skim milk as a reference, according to the formula indicated below [lactose in skim milk - lactose in the sample treated with S. thermophilus I lactose in skim milk] x 100.

[0100] The results obtained show that the exposure of 100 million MaCs not subjected to lyophilisation per ml of skim milk leads to a 27.5% reduction of lactose in the milk in 30 minutes at 37°C.

[0101] It was observed that the exposure of 1 billion MaCs per ml of skim milk leads to 28% reduction of lactose in the milk.

[0102] It is worth noting that, in the absence of metabolic activation, the lactose reduction is about 50% less (see “lyophilised MIM078 MaCs 1 billion / ml” vs “lyophilised MIM078 non-MaCs 1 billion / ml”).

[0103] The quantification of the residual lactose content in a commercial yogurt shows that a standard yogurt production process, which provides for the fermentation of milk for a minimum of 4 hours at 40-42°C, results in a 21% reduction of lactose in the milk.

[0104] Example 3 - Medium acidification ability of MaCs of S. termophilus

[0105] The MaCs of S. thermophilus MIM078 were prepared as described in example 1 and suspended in a saline solution with a final cell density of 1 billion / ml.

[0106] An aliquot (MaCs-Lact) was exposed to lactose 14 mM, whilst a second aliquot (MaCs-Lact-U) was exposed to 14 mM of lactose and 2 mM of urea. MaCs-Lact and MaCs-Lact-U were incubated at 37 °C and the pH of the medium was recorded every 30 s for 30 min.

[0107] The results obtained clearly showed that, in the presence of urea, the MaCs brought about a greater and more rapid acidification of the medium following homolactic fermentation (Figure 1 ).

Claims

CLAIMS1 . An in vitro method for enhancing the ability of urease-positive or ureasenegative bacterial cells to consume lactose, said method comprising at least one step of maintaining the bacterial cells in culture in a solution comprising urea and / or NH3 in the absence of a carbon source, obtaining bacterial cells with an enhanced ability to consume lactose and produce lactic acid.

2. The method according to claim 1 , wherein said method does not envisage the use or addition of antibiotics.

3. The method according to claim 1 or 2, wherein said bacterial cells belong to at least one genus selected from: Streptococcus, Fructilactobacillus, Lactococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus, Pediococcus and combinations thereof.

4. The method according to claim 3, wherein said cells belong to the genus Streptococcus.

5. The method according to claim 4, wherein said cells belong to the species Streptococcus thermophilus.

6. The method according to any one of the preceding claims, wherein the carbon source is lactose.

7. The method according to any one of the preceding claims, wherein said solution comprises a urea concentration between 0.5 mM and 5 mM.

8. The method according to claim 7, wherein said solution comprises a urea concentration between 1 .5 mM and 4 mM.

9. The method according to any one of the preceding claims, wherein the solution comprises a concentration of NH3 between 1 mM and 10 mM.

10. The method according to claim 9, wherein the solution comprises a concentration of NH3 between 3 mM and 8 mM.

11. The method according to any of the preceding claims, wherein the cells are maintained in culture between 15 and 40 minutes.

12. The method according to claim 11 , wherein the cells are maintained in culture between 20 and 35 minutes.

13. Bacterial cells with enhanced lactose-consuming ability obtained by the method according to any one of the preceding claims or a composition comprising them.

14. The bacterial cells according to claim 13 or a composition comprising them for use as a medicament.

15. A food product comprising bacterial cells according to claim 13.

16. The food product according to claim 15 selected from milk and its derivatives selected from: cheese, yogurt, ice cream, even more preferably yogurt and fermented milk or other fermented plant-based drinks (soy milk, oat milk, rice milk).

17. A food supplement comprising bacterial cells according to claim 13.

18. A method for reducing the lactose concentration in a product, comprising at least one step of adding bacterial cells according to claim 13to the product, preferably said product is a food product.

19. The method according to claim 18, wherein the product with added bacterial cells is maintained at a temperature between 35 and 45°C for between 20 and 50 minutes.

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