Fermented dairy product

Abstract

The present invention relates to a fermented dairy product comprising the microorganisms S. thermophilus and L. kefiri,, and which can further comprise a yeast selected from Lachancea sp. and Kluyveromyces lactis. The invention further relates to a method for obtaining said dairy product and food and beverages containing same.

Description

[0001] FERMENTED DAIRY PRODUCT

[0002] FIELD OF INVENTION

[0003] The present invention belongs to the field of food, specifically to the area of ​​dairy products and relates to a fermented dairy product with characteristics similar to yogurt and kefir.

[0004] BACKGROUND

[0005] Yogurt is a dairy product obtained through the fermentation of milk by bacteria belonging to the genera Lactobacillus and Streptococcus. Although different strains can be used, the most common combination is Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0006] These bacteria are capable of transforming lactose into lactic acid through homolactic fermentation, resulting in a decrease in the pH of the medium. The pH range of commercial yogurt is between 4.0 and 4.5. The concentration of organic acids in yogurt can vary depending on several factors, such as the variety of bacteria used, the fermentation time and temperature, and the type of milk used.

[0007] Kefir is another dairy product obtained from the fermentation of milk by microorganisms. It typically uses grains composed of a symbiotic mixture of Levilactobacillus brevis, Lacticaseibacillus paracasei, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactiplantibacillus plantarum, Lentilactobacillus kefiri, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Acetobacter lovaniensis, Acetobacter orientalis, Saccharomyces cerevisiae, Saccharomyces unisporus, Candida kefyr, and Kluyveromyces marxianus. Because some microorganisms present in kefir carry out heterolactic fermentation, in addition to lactic acid, alcohol and CO2 are also produced, which are responsible for its slightly effervescent nature.

[0008] Conventional yogurt is the result of the fermentation of milk by two lactic bacteria; while kefir is a much more complex product with dozens of microorganisms involved, including lactic, acetic and yeasts of various species, which poses a challenge for its standardization and commercial development.

[0009] Kegur is a novel fermented dairy product obtained by fermenting milk with two microorganisms typical of kefir (Lentilactobacillus kefiri) and yogurt (Streptococcus thermophilus), respectively. Numerous prior art documents describe this type of product.

[0010] Document CN107927168A discloses the combination of Lentilactobacillus kefiri KL22 and Streptococcus thermophilus JMCC0019 for obtaining a fermented milk with a good flavor, rich in nutrients, and with a high concentration of microorganisms. It is used as a probiotic with beneficial effects on the gut. This document also discloses a procedure for obtaining this product.

[0011] The scientific article Nambou, K. et al., "A novel approach of direct formulation of defined starter cultures for different kefir-like beverage production," published in the International Dairy Journal, Volume 34, Issue 2, 2014, discloses different combinations of lactic acid bacteria (Lentilactobacillus kefiri and Streptococcus thermophilus) with yeast (Kluyveromyces marxianus). Kluyveromyces marxianus is the sexual form of the Candida kefyr species. However, this document does not disclose any yeast species from the genus Lachancea or the species Kluyveromyces lactis.

[0012] However, none of the prior art documents disclose the combination of Streptococcus thermophilus and Lentilactobacillus kefiri bacteria with the yeasts of the invention to provide the fermented dairy product with different theological and organoleptic properties.

[0013] DESCRIPTION

[0014] In the present invention, the expressions “fermented dairy product”, “Dairy product with Streptococcus thermophilus, Lentilactobacillus kefiri and Lachancea sp.” and “Intense Kegurt” are synonymous and are used interchangeably.

[0015] In the present invention, the expressions “dairy product comprising S. thermophilus, L. kefiri and not comprising Lachancea sp.” and “Soft Kegurt” are synonymous and are used interchangeably.

[0016] In the present invention, the terms “Dairy product consisting essentially of strains of S. thermophilus and L. kefiri” and “Soft kegur” are synonymous and used interchangeably. In this specification, the term “consisting essentially” refers to the fact that the dairy product may include other microorganisms that are common in milk and / or kefir and that do not alter the properties of the final dairy product.

[0017] In the present invention, the terms “dairy product” and “fermented dairy product” are synonymous and used interchangeably. In the present invention, the term “yeast” comprises any species of the genus Lachancea and the species Kluyveromyces lactis.

[0018] A first object of the invention relates to a fermented dairy product comprising the microorganisms S. thermophilus and L. kefiri, the inoculated quantity of L. kefiri being approximately equal to, or slightly greater than, or slightly less than that of S. thermophilus, and not comprising the yeast Lachancea sp. This yeast is not found naturally in dairy products such as kefir or yogurt, but is associated with alcoholic fermentation in wines.

[0019] The term “slightly” means between 1% and 10% by weight of L. kefiri greater or less than the amount of S. thermophilus, or between 2 and 8% by weight, or between 3 and 7% by weight.

[0020] In a particular embodiment, the term “slightly” means between 1% and 10% CFU / ml of L. kefiri higher or lower than the amount of S. thermophilus in CFU / ml. Or between 2 and 8% in CFU / ml, or between 3 and 7% in CFU / ml.

[0021] In a particular embodiment, the term “slightly” means between 1% and 10% of the ODeoo value of L. kefiri being higher or lower than the ODeoo value of S. thermophilus. Or between 2% and 8% ODeoo, or between 3% and 7% ODeoo.

[0022] In one particular embodiment, the fermented dairy product consists essentially of the microorganisms S. thermophilus and L. kefiri, the inoculated amount of L. kefiri being approximately equal to, or slightly greater than, or slightly less than that of S. thermophilus. This dairy product may comprise other microorganisms that are commonly present in milk and / or kefir, but in marginal quantities, for example, less than 10% of the amount of L. kefiri and / or S. thermophilus. preferably less than 10% of the amount of L. kefiri and / or S. thermophilus, more preferably less than 9% of the amount of L. kefiri and / or S. thermophilus, even more preferably less than 8% of the amount of L. kefiri and / or S. thermophilus, or 7%, 6%, 5%, 4%, 3%, 2% or less than 1% of the amount of L. kefiri and S. thermophilus.

[0023] In a particular embodiment, the ratio of inoculated L. kefiri to S. thermophilus is approximately 1:1, or approximately 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2. Or any ratio between approximately 10:1 and approximately 1:10. The ratio may be % by weight, OD600, or CFU / ml.

[0024] The quantity of L. kefiri, S. thermophilus, or any other microorganism in the fermented dairy product can be determined using any standard protocol in the art for quantifying the presence of microorganisms in dairy products. For example, quantitative PCR (qPCR) or colony-forming unit (CFU) counting per unit volume of the product.

[0025] The advantage of this dairy product is that it does not require the addition of sugars and fibers as they are added to the product disclosed in CN107927168A.

[0026] In a particular embodiment, the L. kefiri strain is a strain of L. kefiri deposited in the Spanish Type Culture Collection with number CECT 31007 or strains derived from it.

[0027] This strain was deposited on February 5, 2024 in the Spanish Collection of Type Cultures, Calle Catedrático Agustín Escardino, 9, CP46980, Paterna (Valencia), Spain according to the provisions of the Treaty of Budapest.

[0028] In the present invention, CECT 31007 and CDWN 00937 are synonymous.

[0029] In a particular embodiment, the S. thermophilus strain is a strain of S. thermophilus deposited in the Spanish Type Culture Collection with number CECT 31008 or strains derived from it.

[0030] This strain was deposited on January 25, 2024 in the Spanish Type Culture Collection, Calle Catedrático Agustín Escardino, 9, CP46980, Paterna (Valencia), Spain according to the provisions of the Treaty of Budapest.

[0031] In one particular embodiment, the fermented dairy product comprises the microorganisms S. thermophilus CECT 31008, L. kefiri CECT 31007, the inoculated amount of L. kefiri being approximately equal to, or slightly higher than, or slightly lower than that of S. thermophilus, and does not comprise Lachancea spp.

[0032] In the present invention, CECT 31008 and CDWN 00852 are synonymous.

[0033] In one particular embodiment, the fermented dairy product further comprises a yeast, selected from Lachancea sp. and Kluyveromyces lactis.

[0034] The inventors have surprisingly identified a new fermented dairy product comprising a combination of two lactic acid bacteria: one characteristic of yogurt (S. thermophilus) and the other of kefir (L. kefiri), along with a yeast (Lachancea sp.) that imparts spicy notes similar to those of commercial kefir. This yeast also gives the final fermented dairy product a uniform texture with some bubbles at the bottom, a subtle flavor, and a fresher taste than the combination of the two lactic acid bacteria without the yeast. This result is surprising because, until now, this yeast has only been described in the context of alcoholic beverage production, such as beer. In one particular embodiment, the microorganism Lachancea spp.is one or more of the following species: Lachancea cidri, Lachancea dasiensis, Lachancea fantástica, Lachancea fermentan, Lachancea kluyveri, Lachancea lanzarotensis, Lachancea meyersii, Lachancea mirantina, Lachancea nothofagi, Lachancea quebecensis, Lachancea waltii and Lachancea thermotolerans.

[0035] In another particular embodiment, the Lachancea thermotolerans strain (CDWN 01274) is the strain inoculated for the fermentation process. The Lachancea thermotolerans strain (CDWN 01274) refers to the Lachancea thermotolerans strain deposited in the Spanish Type Culture Collection with number CECT 13233 or strains derived from it.

[0036] This strain was deposited on January 25, 2024 in the Spanish Type Culture Collection, Calle Catedrático Agustín Escardino, 9, CP46980, Paterna (Valencia), Spain according to the provisions of the Treaty of Budapest.

[0037] In the present invention, CECT 13233 and CDWN 01274 are synonymous.

[0038] In another specific embodiment, the fermented dairy product comprises the two lactic acid bacteria described above (S. thermophilus and L. kefir!) and the yeast Kluyveromyces lactis DSMZ 4394. The unbeaten texture is less uniform and has more bubbles, due to the slightly higher carbon dioxide production compared to the combination with Lachancea sp. However, when beaten, the texture closely resembles kefir, becoming creamy and homogeneous. This combination exhibits a higher concentration of acetic acid, which, along with the ethanol, gives it a novel, more intense, somewhat spicy flavor with more acidic notes.

[0039] Surprisingly, the inventors have discovered that K. lactis can be used to obtain the fermented dairy product of the invention, since this strain is primarily used in the prior art for the production of non-alcoholic beers. Furthermore, unlike K. marxianus, K. lactis has the ability to produce lactic acid from lactose; this is advantageous for the production of fermented dairy products, as the higher the lactic acid content, the lower the pH of the product, which is an additional benefit.

[0040] In a preferred embodiment the fermented dairy product comprises the microorganisms S. thermophilus CECT 31008, L. kefiri CECT 31007 and Lachancea thermotolerans CECT 13233.

[0041] A further object of the invention relates to one or more of the following strains CECT 31007, CECT 31008 and CECT13233. A further object of the invention relates to a process for obtaining the fermented dairy product described above.

[0042] The combination of S. thermophilus and L. kefiri is not straightforward, as both species have very different optimal growth temperatures. However, the selection of specific strains (S. thermophilus CDWN 00852 and L. kefiri CDWN 00937) within the diversity of both species, the determination of their compatibility, and the analysis of their growth at different temperatures have allowed the selection of fermentation conditions that permit simultaneous bacterial growth and, therefore, a similar and stable contribution over time to the tertiary and organoleptic properties of the dairy product of the invention.

[0043] The general procedure of the invention comprises inoculating at least one dairy raw material with L. kefiri and S. thermophilus, allowing it to ferment for a suitable period of time and temperature, and then refrigerating the product.

[0044] As dairy raw materials, one can use, for example, milk, whey, fermented milk, lactic bacteria drink, skimmed milk, skimmed milk powder, prepared milk powder, milk powder, concentrated milk, concentrated skimmed milk, condensed milk, condensed skimmed milk, sweetened condensed milk, sweetened condensed skimmed milk, etc.

[0045] In a particular embodiment, the milk may be skimmed, semi-skimmed, whole, or combinations thereof.

[0046] In another particular embodiment, the dairy raw material is obtained from any mammal, such as, for example, cow, goat, sheep, mare, or combinations thereof.

[0047] The quantity of L. kefiri and S. thermophilus inoculated to initiate the procedure of the invention has an optical density, measured at a wavelength of 600 nm (OD6OO), of approximately 0.5 for L. kefiri and approximately 0.1 for S. thermophilus. The microorganisms are grown separately and are preferably inoculated simultaneously, or sequentially as long as they do not alter the tertiary and organoleptic properties of the dairy product of the invention.

[0048] For S. thermophilus, an OD6oo of approximately 0.1 is approximately 1.30 x10 7 CFU / ml (colony-forming units). For L. kefiri, an ODeoo of approximately 0.1 is approximately 9.2 x 10 6 CFU / ml.

[0049] In one particular embodiment, the amount of L. kefiri and S. thermophilus is approximately the same. In another particular embodiment, the amount of L. kefiri is either greater or less than the amount of S. thermophilus. This amount can be measured in either 0Deoo or CFU / ml.

[0050] In another particular embodiment, the ODeoo of L. kefiri is approximately between 0.25 and 0.75 and the ODeoo of S. thermophilus is approximately 0.05 to 0.15. That is, the amount of L. kefiri is approximately between 2.3 x 10 7 ufe / ml at 6.9 x10 7 ufe / mi and the amount of S. thermophilus is approximately between 6.5 x10 6 ufe / ml at 1.95 x10 7 CFU / ml.

[0051] In another particular embodiment, the OD6OO of L. kefiri is approximately between 0.4 and 0.6 and the OD6OO of S. thermophilus is approximately 0.08 to 0.12. That is, the amount of L. kefiri is approximately between 3.68 x10 7 ufe / ml at 5.52 x10 7ufe / mi and the amount of S. thermophilus is approximately between 1.04 x10 7 ufe / ml at 1.56 x10 7 CFU / ml.

[0052] In another particular embodiment, the OD6oo of L. kefiri is approximately 0.5, and the ODeo of S. thermophilus is approximately 0.1. That is, the amount of L. kefiri is approximately 4.67 x 10 7 ufe / mi and the amount of S. thermophilus is approximately between 1.30 x10 7 CFU / ml.

[0053] In one particular embodiment, the ratio of L. kefiri to S. thermophilus is approximately 1:1, or approximately 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2. Or any ratio between approximately 10:1 and approximately 1:10. The ratio of both microorganisms can be determined from ODeoo and / or CFU / ml values ​​and / or % by weight as described in the preceding paragraphs.

[0054] Additionally, Lachancea sp. and / or Kluyveromyces lactis can be added to the product. Inoculation with these microorganisms can be carried out simultaneously with L. kefiri and S. thermophilus, or before or after, but at the appropriate time to ferment the dairy raw material.

[0055] In another particular embodiment, the amount of L. kefiri, S. thermophilus, and Kluyveromyces lactis is approximately the same. The amount is determined from the culture prior to the process of the invention when it reaches an OD6oo of approximately 0.1 for S. thermophilus and Kluyveromyces lactis and an OD6oo of approximately 0.5 for L. kefiri. The amount of feu / ml of L. kefiri and S. thermophilus corresponding to an OD6oo of 0.5 and 0.1, respectively, has been mentioned in the preceding paragraphs. For K. lactis, an OD6oo of approximately 0.1 is approximately 1.16 x 10 5 CFU / ml. The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008.

[0056] In another preferred embodiment, the ODeoo of L. kefiri is approximately 0.25 to 0.75, the ODeoo of S. thermophilus is approximately 0.05 to 0.15, and the ODeoo of K. lactis is approximately 0.05 to 0.75. That is, the amount of L. kefiri is approximately 2.3 x 10 7 ufe / ml at 6.9 x10 7 CFU / ml, the amount of S. thermophilus is approximately between 6.5 x 10 6 ufe / ml at 1.95 x10 7 ufe / mi and the amount of K. lactis is approximately between 5.8 x10 4 ufe / ml at 8.7 x10 5 CFU / ml. The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008,

[0057] In another preferred embodiment, the OD₀ of L. kefiri is approximately 0.4 to 0.6, the OD₀ of S. thermophilus is approximately 0.08 to 0.12, and the OD₀ of K. lactis is approximately 0.05 to 0.6. That is, the amount of L. kefiri is approximately 3.68 x 10 7 ufe / ml at 5.52 x10 7CFU / ml, the amount of S. thermophilus is approximately between 1.04 x 10 7 ufe / ml at 1.56 x10 7 ufe / mi and the amount of K. lactis is approximately between 5.8 x10 4 ufe / ml at 6.96 x10 5 CFU / ml. The L kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008,

[0058] In another preferred embodiment, the OD6oo of L. kefiri is approximately 0.5, the OD6oo of S. thermophilus is approximately 0.1, and the OD6oo of K. lactis is approximately 0.1. That is, the amount of L. kefiri is approximately 4.67 x 10 7 CFU / ml, the amount of S. thermophilus is approximately between 1.30 x 10 7 ufe / mi and the amount of K. lactis is approximately 5.8 x10 5 The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008,

[0059] In another specific embodiment, L. kefiri, S. thermophilus, and K. lactis are added to every 20–300 ml of milk at an ODeoo of between 0.02 and 0.8; preferably, L. kefiri, S. thermophilus, and K. lactis are added to every 50–200 ml of milk at an ODeoo of between 0.03 and 0.7; and more preferably, L. kefiri, S. thermophilus, and K. lactis are added to every 80–150 ml of milk at an ODeoo of between 0.04 and 0.6, the amounts of L. kefiri, S. thermophilus, and Kluyveromyces lactis being approximately the same or different. The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008.

[0060] In another particular embodiment, the quantity of L. kefiri, S. thermophilus, and Lachancea sp. is approximately the same. The quantity is determined from the culture prior to the process of the invention when it reaches an OD600 of 0.1 for S. thermophilus and Lachancea sp. and an OD600 of 0.5 for L. kefiri.

[0061] In a particular embodiment, the microorganism Lachancea sp. is Lachancea thermotolerans, preferably CECT 13233,

[0062] For Lachancea thermotolerans, an OD6oo of approximately 0.1 is approximately 3.73 x10 4 CFU / ml

[0063] In another preferred embodiment, the ODeoo of L. kefiri is approximately 0.25 to 0.75, the ODeoo of S. thermophilus is approximately 0.05 to 0.15, and the ODeoo of Lachancea sp. (preferably Lachancea thermotolerans) is approximately 0.05 to 0.75. That is, the amount of L. kefiri is approximately 2.3 x 10 7 CFU / ml at 6.9 x 10 7 CFU / ml, the amount of S. thermophilus is approximately between 6.5 x 10 6 CFU / ml at 1.95 x 10 7 CFU / ml and the amount of Lachancea sp. (preferably Lachancea thermotolerans) is approximately between 1.87 x10 4 CFU / ml at 2.8 x 10 5CFU / ml. The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008, and / or the L. thermotolerans strain CECT 13233.

[0064] In another preferred embodiment, the OD600 of L. kefiri is approximately 0.4 to 0.6, the OD600 of S. thermophilus is approximately 0.08 to 0.12, and the OD600 of Lachancea sp. (preferably Lachancea thermotolerans) is approximately 0.05 to 0.6. That is, the amount of L. kefiri is approximately 3.68 x 10 7 ufe / ml at 5.52 x10 7 CFU / ml, the amount of S. thermophilus is approximately between 1.04 x 10 7 ufe / ml at 1.56 x10 7 ufe / mi and the amount of Lachancea sp. (preferably Lachancea thermotolerans) is approximately between 1.87 x10 4 ufe / ml at 2.23 x10 5 CFU / ml. The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008, and / or the L. thermotolerans strain CECT 13233.

[0065] In another preferred embodiment, the ODeoo of L. kefiri is approximately 0.5, the ODeoo of S. thermophilus is approximately 0.1, and the ODeoo of Lachancea sp. is approximately 0.1. For Lachancea sp., an ODeoo of approximately 0.1 is approximately 3.73 x 10 4 ufe / mi (preferably Lachancea thermotolerans). That is, the amount of L. kefiri is approximately 4.67 x10 7 , the amount of S. thermophilus is approximately between 1.30 x10 7 ufe / mi and the amount of Lachancea sp. (preferably Lachancea thermotolerans) is approximately 3.73x10 4 CFU / ml. The L. kefiri strain may be CECT 31007 and / or the S. thermophilus strain CECT 31008, and / or the L. thermotolerans strain CECT 13233.

[0066] In another specific embodiment, L. kefiri, S. thermophilus, and Lachancea sp. are added to every 20–300 ml of milk at an OD600 of between 0.02 and 0.8 of each microorganism, preferably to every 50–200 ml of milk at an OD600 of between 0.05 and 0.7, and more preferably to every 80–150 ml of milk at an OD600 of between 0.08 and 0.6 (preferably Lachancea thermotolerans). The L. kefiri strain can be CECT 31007 and / or the S. thermophilus strain CECT 31008, and / or the L. thermotolerans strain CECT 13233.

[0067] An expert in the field would know how to identify the necessary cultivation conditions to obtain the necessary concentration of the microorganisms L. kefiri, S. thermophilus and Lachancea sp. and Kluyveromyces lactis as a preliminary step to putting them in contact with the dairy raw material to carry out the procedure of the invention and obtain the fermented dairy product of the invention.

[0068] Microorganisms can be brought into contact with the dairy raw material simultaneously or one after the other, in any order.

[0069] In a particular embodiment, once the microorganisms come into contact with the dairy raw material, a fermentation process begins. The fermentation process can last from approximately 6 to 50 hours, preferably from 15 to 40 hours, more preferably from approximately 6 to 30 hours, or also approximately 7, 8, 9, 10, 11, 12, 13, 14, or 15 hours. The fermentation process is carried out at a temperature of approximately 15 to 41 °C, preferably from 20 to 41 °C, more preferably from 25 to 40 °C, and more preferably approximately 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 °C.

[0070] In a preferred embodiment, the fermentation temperature is approximately 36.5°C to 38°C as it allows for adequate growth of all microorganisms after 24 hours.

[0071] Once the fermentation process is complete, the dairy product of the invention can be refrigerated, for example, at a temperature in the range of approximately 2 to 10°C, preferably between approximately 3 and 8°C. The technical effect of refrigeration is that it stops the fermentation process and preserves the product until consumption.

[0072] In a preferred embodiment, approximately 100 ml of semi-skimmed milk is inoculated with L. kefiri at an ODeoo of 0.5, S. thermophilus and Lachancea sp. at an ODeoo of 0.1 each. It is left to ferment for approximately 24 hours at approximately 37 °C and then cooled to approximately 4 °C.

[0073] A further object of the invention relates to a fermented dairy product obtained from the process described above. That is, a fermented dairy product characterized in that the product is obtained by fermenting a dairy raw material with a combination of microorganisms comprising S. thermophilus and L. kefiri.

[0074] In one particular embodiment, the fermented dairy product obtained from the process described above comprises a yeast selected from Lachancea sp. and Kluyveromyces lactis. Another object of the invention relates to a food or beverage comprising the fermented dairy product described above.

[0075] The characteristics and advantages of the product of the invention are as follows:

[0076] • Semi-solid (curdled) product like yogurt.

[0077] • Low viscosity like kefir.

[0078] • It contains at least 2 species of microorganisms.

[0079] • Tasting notes and organoleptic properties very positive and similar to yogurt or kefir.

[0080] • Acetic, citric and gluconic acid content similar to kefir and yogurt.

[0081] • Alcohol content less than 1% by volume.

[0082] Additionally, the invention comprises the following clauses:

[0083] 1. Fermented dairy product comprising the microorganisms S. thermophilus and L. kefiri.

[0084] 2. Fermented dairy product according to the previous clause, comprising the microorganisms S. thermophilus, L. kefiri, the ratio of L. kefiri: S. thermophilus being between 10:1 and 1:10.

[0085] 3. Fermented dairy product according to any one of the above clauses, where L. kefiri is strain CECT 31007

[0086] 4. Fermented dairy product according to any one of the above clauses, where

[0087] 5. thermophilus is the CECT 31008 strain

[0088] 5. Fermented dairy product according to any one of the above clauses, not including Lachancea sp.

[0089] 6. Fermented dairy product according to any one of the above clauses, consisting essentially of the microorganisms S. thermophilus and L. kefiri.

[0090] 7. Fermented dairy product according to any one of clauses 1 to 4, comprising the microorganisms S. thermophilus, L. kefiri and a yeast selected from Lachancea sp. and Kluyveromyces lactis.

[0091] 8. Fermented dairy product in accordance with clause 7, comprising the microorganisms S. thermophilus, L. kefiri and Lachancea sp.

[0092] 9. Fermented dairy product according to the preceding clause, wherein the microorganism Lachancea sp. is Lachancea thermotolerans. 10. Fermented dairy product according to the preceding clause, wherein L. thermotolerans is strain CECT 13233.

[0093] 11. Procedure for obtaining the dairy product described in any one of clauses 1 to 6 comprising inoculating L. kefiri and S. thermophilus into at least one dairy raw material, fermenting and then refrigerating.

[0094] 12. Procedure according to the previous clause, where the concentration of L. kefiri, S. thermophilus the amount of L. kefiri is between 2.3 x10 7 ufe / ml at 6.9 x10 7 ufe / mi and the amount of S. thermophilus is between 6.5 x10 6 ufe / ml at 1.95 x10 7 CFU / ml.

[0095] 13. Procedure according to any one of clauses 11 to 12 where L. kefiri is strain CECT 31007 and S. thermophilus is strain CECT 31008

[0096] 14. Procedure according to any one of clauses 11 to 13, where the concentration of L. kefiri: S. thermophilus is between 10:1 and 1:10.

[0097] 15. Procedure for obtaining the dairy product described in any one of clauses 7 to 9 comprising inoculating at least one dairy raw material

[0098] L. kefiri,

[0099] S. thermophilus and

[0100] Lachancea sp. or Kluyveromyces lactis, ferment and then refrigerate.

[0101] 16. Procedure according to the previous clause, where the quantity of L. kefiri is between 2.3 x10 7 ufe / ml at 6.9 x10 7 CFU / ml, the amount of S. thermophilus is between 6.5 x 10 6 ufe / ml at 1.95 x10 7 ufe / mi and the amount of K. lactis is between 5.8 x10 4 ufe / ml at 8.7 x10 5 CFU / ml.

[0102] 17. Procedure according to clause 16, where the quantity of L. kefiri is between 2.3 x10 7 ufe / ml at 6.9 x10 7 CFU / ml, the amount of S. thermophilus is between 6.5 x 10 6 ufe / ml at 1.95 x10 7 ufe / mi and the amount of Lachancea sp. is between 1.87 x10 4 ufe / ml at 2.8 x10 5 CFU / ml.

[0103] 18. A process according to any one of clauses 11 to 17, wherein the dairy raw material is selected from one or more of the following: milk, whey, fermented milk, lactic acid bacteria drink, skimmed milk, skimmed milk powder, prepared milk powder, milk powder, concentrated milk, concentrated skimmed milk, condensed milk, condensed skimmed milk, sweetened condensed milk, and sweetened condensed skimmed milk. 19. A process according to any one of clauses 11 to 18, wherein fermentation occurs between 10 and 50 hours.

[0104] 20. Procedure according to any one of clauses 11 to 19, wherein the fermentation temperature is between 15 and 41 °C

[0105] 21. Procedure according to the previous clause, where the fermentation temperature is between 36.5°C and 38°C.

[0106] 22. Procedure according to any one of clauses 11 to 21, where the cooling temperature is between 2 and 10°C, for a period of time between 24h and 120h.

[0107] 23. Procedure according to any one of clauses 15 to 22 where L. kefiri is strain CECT 31007, S. thermophilus is strain CECT 31008 and L. thermotolerans is strain CECT 13233.

[0108] 24. Fermented dairy product according to any one of clauses 1 to 10, obtained according to the procedure according to any one of clauses 11 to 23.

[0109] 25. Food or beverage comprising the fermented dairy product according to any of clauses 1 to 10 and / or obtained by the process according to any of clauses 11 to 23.

[0110] Each of the terms "comprising," "consisting essentially of," and "consisting of" can be substituted for either of the other two terms. The terms "a" or "an" can refer to one or a plurality of the items they modify (e.g., "a reagent" can mean one or more reagents), unless it is contextually clear that one or more of the items are being described. The term "approximately," as used here, refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%; for example, a weight of "approximately 100 grams" can include a weight between 90 grams and 110 grams). The use of the term "approximately" at the beginning of a list of values ​​modifies each of the values ​​(e.g., "approximately 1, 2, and 3" refers to "approximately 1, approximately 2, and approximately 3").When describing a list of values, the list includes all intermediate values ​​and all fractional values ​​of those values ​​(for example, the list of values ​​"80%, 85%, or 90%" includes the intermediate value 86% and the fractional value 86.4%). When a list of values ​​is followed by the term "or more," the term "or more" applies to each of the listed values ​​(for example, the list "80%, 90%, 95%, or more" or "80%, 90%, or 95% or more" refers to "80% or more, 90% or more, or 95% or more"). When describing a list of values, the list includes all ranges between any two of the listed values ​​(for example, the list "80%, 90%, or 95%" includes the ranges "80% to 90%," "80% to 95%," and "90% to 95%"). Below are some examples of the application of the technology.

[0111] BRIEF DESCRIPTION OF THE FIGURES

[0112] Figure 1: Concentration of lactic acid, acetic acid, gluconic acid, ethanol, citric acid and L-glutamic acid in the dairy product fermented with S. thermophilus and L. kefiri.

[0113] Figure 2: Concentration of lactic acid, acetic acid, gluconic acid, ethanol, citric acid and L-glutamic acid in the dairy product fermented with S. thermophilus, L. kefiri and Lachancea sp.

[0114] Figure 3: Comparison of levels of lactic acid, acetic acid, gluconic acid, ethanol, citric acid and L-glutamic acid in: dairy product with S. thermophilus and L. kefiri (first column), dairy product with S. thermophilus, L. kefiri and Lachancea sp. (second column), kefir (third column) and commercial yogurts (fourth column)

[0115] Figure 4: Growth curve at 34 °C of the three microorganisms used in the production of the dairy product with S. thermophilus, L. kefiri and Lachancea sp.

[0116] Figure 5: Growth curve at 37 °C of the three microorganisms used in the production of the dairy product with S. thermophilus, L. kefiri and Lachancea sp.

[0117] Figure 6: Growth curve at 42 °C of the three microorganisms used in the production of the dairy product with S. thermophilus, L. kefiri and Lachancea sp.

[0118] Figure 7: Ethanol concentration in the different combinations of fermented dairy product: Mild Kegur (dairy product with S. thermophilus and L. kefiri), Intense Kegur 1 (dairy product with S. thermophilus, L. kefiri and Lachancea sp.) and Intense Kegur 2 (dairy product with S. thermophilus, L. kefiri and K. lactis) at three ODeoo values ​​of K. lactis: 0.05; 0.1; and 0.2.

[0119] Figure 8: Acetic acid concentration in the different combinations of fermented dairy product: Mild Kegur (Dairy product with S. thermophilus and L. kefiri), Intense Kegur 1 (Dairy product with S. thermophilus, L. kefiri and Lachancea sp.) and Intense Kegur 2 (Dairy product with S. thermophilus, L. kefiri and K. lactis) at 3 OD6oo of K. lactis: 0.05; 0.1; and 0.2

[0120] EXAMPLES OF IMPLEMENTATION

[0121] 1. Fermented dairy product comprising S. thermophilus and L. kefiri (Soft Kegurt) 1.1 Obtaining

[0122] A sample of the fermented dairy product of the invention was prepared by inoculating 100 ml of semi-skimmed cow's milk with L. kefiri CECT 31007 and S. thermophilus CECT 31008. L. kefiri was inoculated at an ODeoo of 0.5 and S. thermophilus at an ODeoo of 0.1. These ODeoo values ​​correspond to cell density values ​​(cfu / ml) for both bacteria on the order of 10 7The dairy matrix was left to ferment for 24 hours at 37 °C and subsequently refrigerated at 4 °C between 24 and 72 h.

[0123] To obtain the inoculum of the microorganisms used for fermentation, the microorganisms were grown in Petri dishes from inocula obtained from cryopreserved stocks in 15% glycerol at -80 °C. Specifically, the microorganism L. kefiri grows optimally in MRS medium (https: / / www.dsmz.de / microorganisms / medium / pdf / DSMZ_Medium11.pdf) at 30 °C under microaerophilic conditions using the candle jar method for 48 hours. The microorganism S. thermophilus grows at 37 °C in TSA medium (https: / / bacmedia.dsmz.de / medium / 535), and alternatively also in BHI medium (https: / / bacmedia.dsmz.de / medium / 215), under aerobic conditions for 24 hours. Once a triple streak was obtained from each microorganism and the purity and viability of the cultures was verified, a liquid culture was prepared in the same specific medium and the same growth conditions as those specified for the solid media.Once the exponential phase was reached, the biomass of the cultures in liquid medium was concentrated by centrifugation at 12,000 rpm in a Minispin F45-12-11 centrifuge (Eppendorf). The pellet was then washed with saline solution (0.9% NaCl, w / v) to remove any remaining culture medium. Finally, the microorganism inocula were prepared for inoculation into the milk matrix, adjusting the cell densities to the previously specified OD600 values. A BioSpectrometer basic spectrophotometer (Eppendorf) was used to adjust the cell density, setting the final inoculation volumes to 1 ml.

[0124] 2.1 Viscosity

[0125] Viscosity was measured from aliquots of commercial soft kegurt, yogurt, and kefir using an NDJ-1 analog rotational viscometer (COMECTA®) that measures by calculation with a conversion scale (measured value multiplied by the conversion factor). Rotor 1 was used for commercial yogurt; rotor 2 was used for commercial kefir and soft kegurt (Table 1). Table 1: Viscosity values ​​of three fermented dairy products. Values ​​are expressed in centipoise (cp).

[0126] 2. Fermented dairy product comprising S. thermophilus, L. kefiri and Lachancea sp. (Intense Kefiri 1)

[0127] 2.1 Obtaining

[0128] A sample of the dairy product of the invention was prepared by inoculating 100 ml of semi-skimmed cow's milk with L. kefiri strain CECT 31007 at an OD6oo of 0.5, S. thermophilus strain CECT 31008, and Lachancea thermotolerans strain CECT 13233 at an OD6oo of 0.1 each. It was allowed to ferment at 37 °C for 24 hours and then cooled at 4 °C for between 24 and 72 hours.

[0129] The microorganisms were grown analogously to section 1.1, L. thermotolerans in YPD (https: / / bacmedia.dsmz.de / medium / 393) or YM (https: / / bacmedia.dsmz.de / medium / 186) media at 30 °C under aerobic conditions for 24 hours.

[0130] 2.2 pH Measurement

[0131] Once the fermentation process was complete, aliquots were collected to measure the pH of the resulting product and analyze its organoleptic profile. For comparison, a commercial yogurt and kefir, as well as a fermented dairy product containing S. thermophilus and L. kefiri (without the yeast Lachancea sp.), were also analyzed. pH values ​​were measured using a pH meter (PL-700PV, Gondo) and are shown in Table 2. Table 2: pH values ​​obtained from the different combinations of microorganisms and fermented dairy products.

[0132] Dairy products must have a pH between 4 and 4.5 to be suitable for human consumption. Since the pH of the dairy product of the invention is similar to that of other commercial products, the product of the invention is safe for human consumption.

[0133] 2.3 Measurement of metabolites

[0134] The metabolites analyzed to define the organoleptic profile were: lactic acid, acetic acid, gluconic acid, ethanol, citric acid and L-glutamic acid (Figure 1 and Figure 2).

[0135] To determine the profile, 30 ml aliquots were taken from the samples of the products in Table 1 and treated with the Carrez kit “Food Quality” from BioSystems (Ref. 12837), following the manufacturer's instructions.

[0136] The treated samples were analyzed using spectrophotometric methods in an automated instrument (Y15; BioSystems, Barcelona, ​​Spain) that measures the concentration of metabolites from some standard commercial standards: ethanol (BioSystems Ref. 12847), L-lactic acid (BioSystems Ref. 12802), gluconic acid (BioSystems Ref. 12811), L-glutamic acid (BioSystems Ref. 12830), citric acid (BioSystems Ref. 12825) and acetic acid (BioSystems Ref. 12930).

[0137] The organoleptic profiles are very similar in both products. In general, lactic acid is the most abundant organic acid in yogurt and can be present in concentrations ranging from 0.7% to 1.5%. The lactic acid content in kefir is 0.9-1.1%. These data agree with those obtained in the analysis of the dairy product fermented with S. thermophilus, L. kefiri, and Lachancea sp. Acetic acid and citric acid, on the other hand, are found in much lower concentrations, between 0.05% and 0.1%. In the case of ethanol, the dairy product fermented with S. thermophilus, L. kefiri, and Lachancea sp. has a slightly higher content compared to commercial kefir, which has an alcohol percentage between 0.3% and 1.0%. This could be due to the presence of L. kefiri and Lachancea sp. They produce ethanol during fermentation.

[0138] Figure 3 shows a comparison of the organoleptic profile between mild and intense Kegur, and commercial kefir and yogurt. Specific values ​​are shown in Table 3.

[0139] Table 3 Organoleptic data of fermented dairy products

[0140] 2.3 Determination of the optimum fermentation temperature

[0141] The growth curves of the three strains involved in the fermentation process were analyzed at different incubation temperatures, specifically 34 °C, 37 °C, and 42 °C, to determine their growth dynamics and optimum. This analysis allowed for the definition of a consensus optimum temperature for the microorganisms, enabling the simultaneous incubation of all three, thus simplifying the process.

[0142] To obtain the growth curves at 37 °C of the three microorganisms used in the preparation of the fermented dairy product of the invention, the following procedure was carried out:

[0143] From a solid-mediated culture of each microorganism, the biomass collected with an inoculating loop was resuspended in 100 ml of phosphate-buffered saline (PBS), and the ODeoo was subsequently measured using a spectrophotometer. In a 1.5 ml Eppendorf tube, a suspension was prepared in a culture medium specific to each strain to adjust the ODeoo to a final value of 1 in a final volume of 100 ml of culture medium specific to each strain. For L. kefiri, MRS medium supplemented with 5% (w / v) yeast extract was used; for S. thermophilus, TSB medium; and for L. thermotolerans, YPD medium.

[0144] In a 96-well, flat-bottomed, transparent SBS plate, appropriate dilutions were prepared for measuring growth curves. Two negative controls were included for each culture medium (200 µL per well). A 1 / 10 dilution was prepared for each strain (180 µL of culture medium and 20 µL of microbial suspension adjusted to an OD6‰ of 1). All measurements were performed in duplicate (biological replicates). The multiwell plates were incubated in an Infinite M nano+ plate reader (Tecan) at the selected assay temperature for 48 hours, measuring the OD6‰ at 30-minute intervals for 96 reading cycles. Although L. kefiri showed better growth at 34 °C (Figure 4), S. thermophilus grew poorly and more slowly at that temperature.For this reason, 37°C was chosen as the optimal temperature for the fermentation process, as it allows for the adequate growth of all microorganisms involved in the process after 24 hours of incubation (Figure 5).

[0145] 3. Fermented dairy product comprising S. thermophilus, L. kefiri and Kluyveromyces lactis (Intense Kegur 2)

[0146] 3.1 Obtaining

[0147] Three samples of the dairy product of the invention were prepared by inoculating 100 ml of semi-skimmed cow's milk with L. kefiri (OD600 0.5), S. thermophilus (OD600 0.1) and K. lactis at an OD600 of 0.05, 0.1 and 0.2. It was allowed to ferment for 24 hours at 37 °C and then cooled at 4 °C for 72 h. The procedure of section 2.1 was followed except for the differences indicated in this section, for example, Kluyveromyces lactis was grown in YPD or YM medium at 30 °C for 24 hours.

[0148] 3.2 Measurement of metabolites

[0149] The concentrations of ethanol (Fig. 7) and acetic acid (Fig. 8) were measured in the fermented dairy product described in section 2.2 and compared with the fermented dairy product comprising S. thermophilus, L. kefiri, and Lachancea sp., and with soft Kegur. These products were obtained as described in section 1.1, and the metabolites were measured using the same protocol as described in section 1.3. It was observed that the dairy product fermented with K. lactis produced higher levels of ethanol and acetic acid than the other products, with the concentrations of both metabolites increasing as the OD600 of K. lactis increased. Therefore, the two products, intense kegur 1 (S. thermophilus, L. kefiri and Lachancea sp.) and intense kegur 2 (S. thermophilus, L. kefiri and K. lactis) are different, since they have different composition and organoleptic characteristics.

Claims

CLAIMS 1. Fermented dairy product comprising the microorganisms S. thermophilus, L. kefiri and a yeast selected from Lachancea sp. and Kluyveromyces lactis.

2. Fermented dairy product according to the preceding claim, comprising the microorganisms S. thermophilus, L. kefiri and Lachancea sp.

3. Fermented dairy product according to the preceding claim wherein the microorganism Lachancea sp. is Lachancea thermotolerans.

4. Fermented dairy product comprising the microorganisms S. thermophilus and L. kefiri.

5. Fermented dairy product according to the preceding claim, comprising the microorganisms S. thermophilus, L. kefiri, the ratio of L. kefiri: S. thermophilus being between 10:1 and 1:

10.

6. Fermented dairy product according to any one of the preceding claims, not comprising Lachancea sp.

7. Fermented dairy product according to any one of the preceding claims, consisting essentially of the microorganisms S. thermophilus and L. kefiri.

8. Fermented dairy product according to any one of the preceding claims, wherein L. kefiri is strain CECT 31007 9. Fermented dairy product according to any one of the preceding claims, wherein S. thermophilus is strain CECT 31008 10. Fermented dairy product according to any one of the preceding claims, wherein L, thermotolerans is the CECT 13233 strain 11. A method for obtaining the dairy product described in any one of claims 4 to 9 comprising inoculating L. kefiri and S. thermophilus into at least one dairy raw material, fermenting, and then refrigerating.

12. Process according to the preceding claim, wherein the concentration of L. kefiri is between 2.3 x10 7 ufe / ml at 6.9 x107 ufe / mi and the concentration of S. thermophilus is between 6.5 x10 6 ufe / ml at 1.95 x10 7 CFU / ml.

13. Method according to any one of claims 11 to 12 wherein L. kefiri is strain CECT 31007 and S. thermophilus is strain CECT 31008 14. Process according to any one of claims 11 to 13, wherein the concentration of L. kefiri: S. thermophilus is between 10:1 and 1:

10.

15. A method for obtaining the dairy product described in any one of claims 1 to 3 comprising inoculating at least one dairy raw material L. kefiri, S. thermophilus and Lachancea sp. or Kluyveromyces lactis, ferment and then refrigerate.

16. Method according to the preceding claim, wherein the amount of L. kefiri is between 2.3 x10 7 ufe / ml at 6.9 x10 7 CFU / ml, the amount of S. thermophilus is between 6.5 x 10 6ufe / ml at 1.95 x10 7 ufe / mi and the amount of K. lactis is between 5.8 x10 4 ufe / ml at 8.7 x10 5 CFU / ml.

17. Method according to claim 16, wherein the amount of L. kefiri is between 2.3 x10 7 ufe / ml at 6.9 x10 7 CFU / ml, the amount of S. thermophilus is between 6.5 x 10 6 ufe / ml at 1.95 x10 7 ufe / mi and the amount of Lachancea sp. is between 1.87 x10 4 ufe / mi a 2.8 x10 5 CFU / ml.

18. A process according to any one of claims 11 to 17, wherein the dairy raw material is milk 19. Process according to any one of claims 11 to 18, wherein the fermentation occurs between 10 and 50 h.

20. A process according to any one of claims 11 to 19, wherein the fermentation temperature is between 15 and 41°C 21. Process according to the preceding claim, wherein the fermentation temperature is between 36.5°C and 38°C.

22. Method according to any one of claims 11 to 21, wherein the cooling temperature is between 2 and 10°C, for a period of time between 24h and 120h.

23. Method according to any one of claims 15 to 22 wherein L. kefiri is strain CECT 31007, S. thermophilus is strain CECT 31008 and L. thermotolerans is strain CECT 13233.

24. Fermented dairy product according to any one of claims 1 to 10, obtained according to the process according to any one of claims 11 to 23.

25. Food or beverage comprising the fermented dairy product according to any one of claims 1 to 10 and / or obtained by the process according to any one of claims 11 to 23.

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