Compositions, methods for preparing same and uses

Abstract

A composition, its preparation method and use are provided. The composition comprises, as raw materials, 2.5% to 80% by weight of inactivated Kluyveromyces yeast cells, 1% to 50% by weight of edible oil, and 18% to 96.5% by weight of water. The composition is stable and homogeneous, and is rich in nutrients such as protein and dietary fiber, and can be used in the development of beverage and food ingredients, and has nutritional, economical and sustainable properties.

Description

[Technical Field]

[0001] The present disclosure is in the field of food processing, and specifically relates to compositions, methods for their preparation and uses. [Background technology]

[0002] Kluyveromyces yeast is a type of ascosporogenous yeast and is food-safe. Among them, Kluyveromyces marxianus and Kluyveromyces lactis are widely used industrially and are the most studied yeasts. For example, Kluyveromyces marxianus is widely found in yogurt, fruit, and kefir. Due to its high food safety, high growth rate, high biomass, and high temperature tolerance, it is widely used in fermentation and lactic acid bacteria beverage production. However, the nutritional and physicochemical properties of inactivated Kluyveromyces yeast itself have not been fully studied.

[0003] Emulsification is currently the most common processing method in the food industry, especially in beverage and sauce production. To achieve a uniform, stable, and non-stratified product, emulsification typically involves uniformly mixing the aqueous and oil phases and adding a combination of one or more emulsifiers in a certain amount. This prevents heterogeneous phases from repelling each other and homogeneous phases from flocculating, ensuring long-term system stability and product shelf life. Emulsifiers are classified as surfactants in the chemical industry and food additives in the food industry. There are three types of emulsifiers commonly used in beverage and sauce processing: ionic, nonionic, and ampholyte. The properties and functions of these three types of emulsifiers are typically determined by the relative strength of the hydrophilic and lipophilic groups within the molecule. A good emulsifier system must have a significant balance between hydrophilic and hydrophobic groups. Existing food processing technologies employ emulsifiers to achieve stable, uniform emulsions, which typically require rigorous theoretical calculations to balance the hydrophilic and hydrophobic groups within the emulsion. Due to the diversification of food formulations, achieving a relatively stable formulation typically requires theory, experience, numerous experiments, or simply excessive use of emulsifiers. Therefore, research into natural and simple processes for the long-term stability and uniformity of foods containing both aqueous and oil phase components is an urgent issue in the industry. Summary of the Invention [Problem to be solved by the invention]

[0004] The problem to be solved by the present disclosure is to provide a food composition that does not contain food additives, is stable and uniform, and is high in nutrients such as protein and dietary fiber.

[0005] The present disclosure also provides the use of the food composition in the preparation of a food product. [Means for solving the problem]

[0006] To achieve the above objectives, the present disclosure provides the following solutions:

[0007] In one embodiment of the present disclosure, a composition is provided containing, as raw materials, 2.5% to 80% by weight of the inactivated Kluyveromyces yeast cells described above, 1% to 50% by weight of an edible oil, and 18% to 96.5% by weight of water. Research in the present disclosure has shown that the composition and proportions of this raw material are conducive to the formation of a stable and uniform emulsion system. The inactivated Kluyveromyces yeast cells can physically isolate oil particles from each other and can be directly applied to the emulsification process of an aqueous phase and an oil phase in the absence of exogenous food additives such as emulsifiers, stabilizers, and thickeners, resulting in a stable and uniform emulsion system that can be maintained for at least 7 days without emulsion breakdown or stratification.

[0008] In one embodiment of the present disclosure, the content of the inactivated Kluyveromyces yeast cells is 10 to 70 wt %.

[0009] In one embodiment of the present disclosure, the content of the edible oil is 10 to 40% by weight.

[0010] In one embodiment of the present disclosure, the composition may contain, as ingredients, 2.5 to 25 wt% inactivated Kluyveromyces yeast cells, 1 to 40 wt% edible oil, and 50 to 96.5 wt% water, e.g., 2.5 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, or 25 wt% inactivated Kluyveromyces cells and 1 wt%, 2 wt%, 4 wt%, 8 wt%, 12 wt%, 16 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, or 40 wt% edible oil. This ingredient composition and proportions help maintain emulsion for at least 28 days without demulsification or stratification.

[0011] In an embodiment of the present disclosure, when the content of inactivated Kluyveromyces yeast cells is 2.5 to 25%, The frequency of components with a particle size of ≦5 μm is 5 to 98%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%. The frequency of components with a particle size of ≦10 μm is 8 to 100%, and can be, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The frequency of components with a particle size of ≦50 μm is 38 to 100%, and can be, for example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The frequency of components with particle diameters of ≦100 μm is approximately 100%. This frequency distribution helps maintain the long-term stability of this composition system.

[0012] In one preferred embodiment of the present disclosure, the composition contains 30 to 70% by weight of inactivated Kluyveromyces yeast cells, 1 to 30% by weight of edible oil, and 18 to 69% by weight of water as ingredients, and may contain, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight of inactivated Kluyveromyces yeast cells and 1%, 2%, 4%, 8%, 12%, 16%, 20%, 25%, or 30% by weight of edible oil. This composition and ratio of ingredients can be maintained for at least 28 days without demulsification or stratification.

[0013] In an embodiment of the present disclosure, when the content of inactivated Kluyveromyces yeast cells is 30 to 70%, The frequency of components with a particle size of ≦5 μm is 54 to 100%, and can be, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. The frequency of components with a particle size of ≦10 μm is 55 to 100%, and can be, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. The frequency of components with a particle size of ≦50 μm is 75 to 100%, and can be, for example, 75%, 80%, 85%, 90%, or 95%. The frequency of components with particle diameters of ≦100 μm is approximately 100%. This frequency distribution helps maintain this composition system for at least 28 days without demulsification or stratification.

[0014] According to the present disclosure, the particle size of the inactivated Kluyveromyces yeast cells in the composition can be 1 to 7 μm, for example, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 7 μm, which allows them to be used as a "caulking agent" to separate oil particles from each other in oil-in-water systems (where the proportion of water is greater than the proportion of oil), or to form microparticles in water to separate oil particles from each other through a polar effect in water-in-oil systems (where the proportion of oil is greater than the proportion of water), thereby delaying the demulsification time of the composition system.

[0015] In one preferred embodiment of the present disclosure, the composition is an oil-in-water system.

[0016] Specifically, the inactivated Kluyveromyces yeast cells are one or more species selected from the group consisting of Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickerhamii, and Kluyveromyces thermotolerans. According to the present disclosure, selecting these five types of yeast cells is more preferable for obtaining inactivated Kluyveromyces yeast cells within the above particle size range.

[0017] Preferably, the inactivated Kluyveromyces yeast cells are Kluyveromyces marxianus and / or Kluyveromyces lactis cells, where Kluyveromyces marxianus is an edible fungus species designated by the National Health Commission, and Kluyveromyces lactis cells are a fungus species designated by the National Health Commission as being usable in health foods.

[0018] In one preferred embodiment of the present disclosure, the cell particle size of the inactivated Kluyveromyces yeast cells in the composition may be 2 to 5 μm, which is advantageous in a composition system in which an aqueous phase component and an oil phase component coexist, in that the oil particles are isolated from each other so as to prevent contact with each other and delay the time required for emulsification to break.

[0019] According to further studies of the present disclosure, when the inactivated Kluyveromyces yeast cells are Kluyveromyces hubeiensis cells, the particle size thereof may be 2 to 5 μm, for example, 2 μm, 3 μm, 4 μm, or 5 μm.

[0020] When the inactivated Kluyveromyces yeast cells are Kluyveromyces wickerhamii cells, the particle size may be 2 to 7 μm, for example, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 7 μm.

[0021] When the inactivated Kluyveromyces yeast cells are Kluyveromyces thermotolerans cells, the particle size may be 2 to 5 μm, for example, 2 μm, 3 μm, 4 μm, or 5 μm.

[0022] When the inactivated Kluyveromyces yeast cells are Kluyveromyces marxianus cells, the particle size may be 2 to 7 μm, for example, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 7 μm.

[0023] When the inactivated yeast cells of the genus Kluyveromyces are Kluyveromyces lactis cells, the particle size may be 2 to 7 μm, for example, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 7 μm.

[0024] According to the research of the present disclosure, the particle size of the edible oil in the composition can be 1 to 100 μm, and controlling the particle size of the edible oil in this composition within the range of 1 to 100 μm is more conducive to achieving a balance between processing technology and product stability. If the particle size of the edible oil in the composition is too large, it becomes difficult for the inactivated Kluyveromyces yeast cells to play an effective physical isolation role within the composition, and the oil particles rapidly combine to form large oil droplets, which in turn causes emulsification and stratification of the system and affects product stability.

[0025] In one specific embodiment of the present disclosure, the particle size of the edible oil in the composition is 5 to 100 μm, and may further be 5 to 10 μm, 5 to 50 μm, 10 to 50 μm, 10 to 100 μm, or 50 to 100 μm, for example, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, or 95 μm.

[0026] The particle size of the edible oil in this composition is calculated from the average particle size of the composition in the system, the average particle size of the yeast cells, and the frequency of the edible oil particle size. The average particle size of the yeast cells in the composition, the proportion of each component, and the total frequency of the particle size distribution of the system are known, and the frequency of the particle size of the edible oil = total frequency of the particle size distribution of the system - frequency of the average particle size of the yeast cells.

[0027] Specifically, the inactivated Kluyveromyces yeast cells described above contain 25-55% protein, 1-5% fat, and 15-30% dietary fiber. Because they are high in protein, low in fat (including saturated and trans fatty acids), and rich in water-soluble dietary fiber, they can be used as a nutrient source for food applications.

[0028] In one preferred embodiment of the present disclosure, the inactivated Kluyveromyces yeast cells contain 30.5 to 52.5% protein, 2.1 to 4.8% fat, and 15.5 to 29.0% dietary fiber.

[0029] Specifically, the inactivated Kluyveromyces yeast cells have a moisture content of 6 to 10% and an ash content of 2.5 to 10.5%.

[0030] In one embodiment of the present disclosure, the inactivated Kluyveromyces yeast cells may be dried inactivated Kluyveromyces yeast cells (inactivated Kluyveromyces yeast cell dry powder), dehydrated inactivated Kluyveromyces yeast cells, or a suspension of inactivated Kluyveromyces yeast cells.

[0031] In one preferred embodiment of the present disclosure, the inactivated Kluyveromyces yeast cells are an inactivated Kluyveromyces yeast cell suspension.

[0032] In one preferred embodiment of the present disclosure, the inactivated Kluyveromyces yeast cells are dried inactivated Kluyveromyces yeast cells, which are more convenient for preservation, transportation, storage, and use of the composition.

[0033] In one embodiment of the present disclosure, the edible oil described herein is one or more selected from soybean oil, rapeseed oil, high oleic sunflower oil, medium chain triglyceride, canola oil, coconut oil, corn oil, sesame oil, tea seed oil, rice bran oil, olive oil, linseed oil, safflower oil, grapeseed oil, walnut oil, palm oil, peanut oil, and blended oils.

[0034] In one preferred embodiment of the present disclosure, the edible oil described herein is one or more selected from coconut oil, medium chain triglycerides, canola oil, high oleic sunflower oil, olive oil, and rapeseed oil.

[0035] The inactivated Kluyveromyces yeast cells are (1) selecting a medium containing a carbon source, a nitrogen source, and salts, and culturing a yeast strain of the genus Kluyveromyces for 15 to 40 hours; and (2) adjusting the pH of the medium to 4.0 to 8.0, fermenting at 25 to 50°C, and then inactivating the medium by heating to obtain the inactivated Kluyveromyces yeast cells.

[0036] Specifically, the medium containing a carbon source, a nitrogen source, and salts may be a medium obtained by combining one or more carbon sources, nitrogen sources, and salts selected from molasses, glucose, starch, dipotassium hydrogen phosphate, dry corn syrup, sodium hydroxide, magnesium sulfate, ammonium sulfate, aqueous ammonia, urea, sodium chloride, yeast extract, peptone, potassium dihydrogen phosphate, potassium hydroxide, methionine, cysteine, alanine, glycine, and glutamic acid, but is not limited to these.

[0037] In one embodiment of the present disclosure, the medium containing a carbon source, a nitrogen source, and salts may be a medium containing glucose, magnesium sulfate, ammonium sulfate, yeast extract, and potassium dihydrogen phosphate, and specifically, the weight percentages of each component are 3.5-4.5% glucose, 0.2-7% molasses, 0.1-0.3% dry corn syrup, 0.02-0.15% magnesium sulfate, 0.5-0.6% ammonium sulfate, 0.6-0.9% yeast extract, and 0.4-0.6% potassium dihydrogen phosphate. The medium may also contain other inorganic salt components such as copper sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, and zinc sulfate, as well as nutrients such as methionine, alanine, cysteine, and glycine, with the remainder being water.

[0038] Specifically, the culture time of the yeast strain of the genus Kluyveromyces in the medium is 15 to 40 hours, and may be, for example, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, or 40 hours.

[0039] Specifically, the pH of the medium may be 4.0 to 8.0, for example, 4, 4.5, 5.0, 5.5, 6, 6.5, 7, 7.5, or 8.

[0040] Specifically, the fermentation temperature may be 25 to 50°C, for example, 25 to 30°C, 25 to 35°C, 25 to 40°C, 25 to 45°C, 30 to 35°C, 30 to 40°C, 30 to 45°C, 30 to 50°C, 35 to 40°C, 35 to 45°C, 35 to 50°C, 40 to 45°C, or 45 to 50°C.

[0041] Specifically, the fermentation time may be 15 to 40 hours, for example, 15 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 36 hours, or 40 hours.

[0042] In one embodiment of the present disclosure, the above-mentioned dried inactivated Kluyveromyces yeast cells, dehydrated inactivated Kluyveromyces yeast cells, and suspension of inactivated Kluyveromyces yeast cells are prepared by the following method. A medium containing carbon, nitrogen, and salts is selected, Kluyveromyces yeast cells are cultured for 15 to 40 hours, the pH of the medium is adjusted to 4.0 to 8.0, and fermentation is carried out at 25 to 50°C for 15 to 40 hours. The culture is then inactivated by heating, centrifuged, and the supernatant is removed to obtain a Kluyveromyces yeast cell slurry. The Kluyveromyces yeast cell slurry is then post-treated to obtain a treated bacterial suspension, which is an inactivated Kluyveromyces yeast cell suspension.

[0043] The treated bacterial suspension is then dried to obtain dried inactivated Kluyveromyces yeast cells, and dehydrated to obtain dehydrated inactivated Kluyveromyces yeast cells.

[0044] In an embodiment of the present disclosure, the drying treatment is a drying technique commonly used in the field, such as freeze drying, spray drying, or bake drying, and the dehydration treatment is a dehydration technique commonly used in the field, and the present disclosure is not particularly limited thereto.

[0045] Specifically, the heat inactivation is carried out at 100°C, and the heat inactivation time may be 20 minutes or more, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, or 40 minutes.

[0046] The post-treatment process may include one or more selected from water washing, pH adjustment, alcohol precipitation, alcohol extraction, activated carbon adsorption, ozone treatment, protease treatment, cellulase treatment, hemicellulase treatment, lipase treatment, freezing treatment, solution pressure treatment, and heat treatment. The post-treatment process is a process commonly used in the art, and the present disclosure does not particularly limit the scope thereof.

[0047] The protease may be an endopeptidase or exopeptidase derived from a microorganism, a plant, or an animal, such as a serine protease, a cysteine ​​protease, an aspartic acid protease, a microorganism-derived protease, a plant-derived papain or pineapple protease, or an animal-derived trypsin, pepsin, or histone. The cellulase is an enzyme capable of decomposing cellulose to produce glucose, such as β-1,4-glucan-4-glucanohydrolase.

[0048] A further aspect of the present disclosure is (1) mixing inactivated Kluyveromyces yeast cells with water to form a cell suspension; and (2) adding an edible oil during shear emulsification of the suspension and continuously shear-emulsifying the edible oil until the particle size of the edible oil reaches 1 to 100 μm.

[0049] In one embodiment of the present disclosure, the inactivated Kluyveromyces yeast cells are an inactivated Kluyveromyces yeast cell suspension or an inactivated Kluyveromyces yeast cell dry powder.

[0050] The proportion of the inactivated Kluyveromyces yeast cells in the composition described in the present disclosure is not affected by the form of the inactivated Kluyveromyces yeast cells added during the preparation process. Naturally, when the inactivated Kluyveromyces yeast cells used are inactivated Kluyveromyces yeast cell dry powder, they can be added directly according to the above-mentioned proportion. When the inactivated Kluyveromyces yeast cells used are inactivated Kluyveromyces yeast cell suspension, the proportion of the inactivated Kluyveromyces yeast cells in the composition can be set to the above-mentioned proportion by calculating the cell number.

[0051] A further aspect of the present disclosure provides a food product comprising the composition.

[0052] In one embodiment of the present disclosure, when the content of inactivated Kluyveromyces yeast cells in the composition is 2.5 to 25%, the composition is a beverage, and the beverage composition comprises 2.5 to 25% inactivated Kluyveromyces yeast cells, 1 to 40% edible oil, and 40 to 96.5% water.

[0053] In one embodiment of the present disclosure, when the content of inactivated Kluyveromyces yeast cells in a composition is 30 to 70%, the composition is a sauce, and the sauce composition contains 30 to 70% inactivated Kluyveromyces yeast cells, 1 to 30% edible oil, and 18 to 69% water.

[0054] A further aspect of the present disclosure provides the use of the composition in the preparation of a food product.

[0055] In one embodiment of the present disclosure, the composition is preferably used to prepare biscuits, bread, baked foods, puffed foods, freeze-dried foods, ice cream, and dehydrated foods, and the composition described in the present disclosure has a high protein content and can be added to foods as a protein substitute. At the same time, the food composition described in the present disclosure has properties including low fat (saturated fat, trans fat), abundant water-soluble dietary fiber, etc., and has superior food value.

[0056] In an embodiment of the present disclosure, the food composition beverage is added in an amount greater than 10% when used in the preparation of a food product, and the food composition sauce is added in an amount greater than 3% when used in the preparation of a food product. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of the inactivated Kluyveromyces hubeiensis having the stabilizing effect of Example 1, taken by a scanning electron microscope. [Figure 2] FIG. 1 is a schematic diagram of inactivated Kluyveromyces wickerhamii having the stabilizing effect of Example 1, taken by a scanning electron microscope. [Figure 3] FIG. 1 is a schematic diagram of the inactivated Kluyveromyces thermotolerans having the stabilizing effect of Example 1, taken by a scanning electron microscope. [Figure 4] FIG. 1 is a schematic diagram of the inactivated Kluyveromyces marxlanus having the stabilizing effect of Example 1 taken by a scanning electron microscope. [Figure 5] FIG. 1 is a schematic diagram of the inactivated Kluyveromyces lactis having the stabilizing effect of Example 1, taken by a scanning electron microscope. [Figure 6] 1 shows microscopic photographs of compositions of Example 8 with different blending ratios. DETAILED DESCRIPTION OF THE INVENTION

[0058] In order to more clearly describe the technical solutions in the embodiments of the present disclosure or the prior art, the following briefly describes the accompanying drawings used in the description of the embodiments or the prior art. However, the accompanying drawings in the following description are only one embodiment of the present disclosure, and it is obvious to those skilled in the art that other embodiments can be obtained according to these drawings.

[0059] I. Definition In the present disclosure, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art unless otherwise specified.In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, chemical-related terms and laboratory operation steps used herein are terms and common procedures widely used in the corresponding fields.In addition, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0060] Although, for clarity and conciseness of description, features are described herein as part of several identical or separate embodiments, it will be understood that the scope of the disclosure may encompass multiple embodiments having combinations of all or some of the described features.

[0061] As used herein, unless otherwise specified, the term "about" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0062] As used herein, unless otherwise indicated, the terms "comprise," "have," and "contain," including their grammatical equivalents, are generally to be understood as open-ended and non-limiting, e.g., not excluding other elements or steps not listed.

[0063] The term "fermentation" as used herein refers to the process of converting external substrates using specific metabolic pathways within living cells under appropriate conditions to produce a desired product or organism required by humans.

[0064] The term "shear emulsification" refers to the process of using centrifugal force generated by a rotor that rotates at high speed and power to radially feed ingredients into the narrow, precise gap between the stator and rotor, and then subjecting them to forces such as centrifugal compression or impact, or by adding high-pressure homogenization at a later stage, to uniformly disperse, mix, and emulsify the ingredients.

[0065] The term "food additive" means a synthetic or natural substance added to food to improve its quality, color, aroma, or taste, or for the purposes of preservation, freshness, or processing. It also includes food flavorings, bases in gum-based confectioneries, and processing aids for the food industry.

[0066] The term "drying" refers to the process of using heat to evaporate the water in a wet material and then using air currents or vacuum to remove the evaporated water, resulting in a dry material.

[0067] II. MODE FOR CARRYING OUT THE INVENTION The Kluyveromyces yeast CJ3113 used in the embodiments of the present disclosure is preserved at the China Typical Culture Preservation Center (CCTCC) of the Wuhan University Preservation Center, Wuhan City, Hubei Province, under the preservation number CCTCC No: M20211265, has the Latin scientific name Kluyveromyces marxianus, and was preserved on October 13, 2021.

[0068] Kluyveromyces lactis was purchased from the China Center for Industrial Microbial Cultures (CIMSC) (Culture No. CICC 32428).

[0069] Kluyveromyces hubeiensis, Kluyveromyces wickerhamii, and Kluyveromyces thermotolerans were purchased from the Comprehensive Microbiology Center of the China Committee for the Preservation of Microorganisms (CGMCC), with preservation numbers CGMCC 2.4330, CGMCC 2.4309, and CGMCC 2.4072, respectively.

[0070] Example Example 1: Measurement of particle size on which Kluyveromyces yeast has a stabilizing effect The minimum and maximum particle sizes (μm) of the stabilizing inactivated Kluyveromyces marxianus, inactivated Kluyveromyces lactis, inactivated Kluyveromyces hubeiensis, inactivated Kluyveromyces wickerhamii, and inactivated Kluyveromyces thermotolerans were observed and measured using a scanning electron microscope (ZEISS GeminiSEM500, field emission). See Table 1 for details. Schematic diagrams of the scanning electron microscope images are shown in Figures 1–5.

[0071] Table 1. Minimum and maximum particle sizes for which Kluyveromyces yeast has a stabilizing effect [Table 1]

[0072] Example 2: Preparation of inactivated Kluyveromyces hubeiensis cell dry powder Kluyveromyces hubeiensis was cultured in a medium containing carbon, nitrogen, and salt sources (4.5% glucose, 0.2% molasses, 0.2% dry corn syrup, 0.02% magnesium sulfate, 0.6% ammonium sulfate, 0.6% yeast extract, 0.6% potassium dihydrogen phosphate, 6 ppm copper sulfate, 12 ppm ferrous sulfate, 15 ppm manganese sulfate, 6 ppm cobalt chloride, 20 ppm zinc sulfate, 8 ppm methionine, 5 ppm alanine, 2 ppm cysteine, and 5 ppm glycine). The pH of the medium was adjusted to 4.0-5.0, and the mixture was fermented at 25-30°C for 40 hours. The mixture was then heat-inactivated at 100°C for 20 minutes, centrifuged, and the supernatant was removed to obtain a Kluyveromyces yeast suspension. The Kluyveromyces yeast suspension was then subjected to the following post-treatment. The bacterial suspension was centrifuged, soaked in deionized water (5x volume) for 30 minutes, adjusted to pH 5.0 with food-grade lactic acid, and incubated at 50°C for 12 hours with 0.01% acid protease and 0.01% papain. The bacterial suspension was then incubated at 50°C for 12 hours and the particle size was measured using a laser particle size analyzer. The pH was adjusted to 6.6-6.8 with food-grade sodium bicarbonate, and the bacterial suspension was incubated at 55°C for 36 hours with 0.02% β-glucanase and 0.01% alkaline protease. The bacterial suspension was then decolorized by activated carbon adsorption to obtain an inactivated Kluyveromyces yeast cell suspension. This suspension was then frozen at -20°C for 1 hour, cooled, and sterilized by immersion in 85% ethanol (3x volume) for 60 minutes. After recovering the ethanol, the bacterial suspension was mixed with deionized water in an amount equal to the weight of the bacterial suspension. Ozone was introduced into the solution for 1 hour for sterilization, and the solution was then pasteurized or autoclaved, followed by spray drying to obtain a dry powder of inactivated Kluyveromyces hubeiensis cells.

[0073] Example 3: Preparation of inactivated Kluyveromyces wickerhamii cell dry powder Kluyveromyces wickerhamii was cultured in a medium containing carbon, nitrogen, and salts (4.2% glucose, 0.5% molasses, 0.1% dry corn syrup, 0.03% magnesium sulfate, 0.5% ammonium sulfate, 0.6% yeast extract, 0.4% potassium dihydrogen phosphate, 5 ppm copper sulfate, 10 ppm ferrous sulfate, 10 ppm manganese sulfate, 5 ppm cobalt chloride, 18 ppm zinc sulfate, 5 ppm methionine, 6 ppm alanine, 3 ppm cysteine, and 1 ppm glycine). The pH of the medium was adjusted to 4.0-5.0, and the mixture was fermented at 45-50°C for 15 hours. The mixture was then heat-inactivated at 100°C for 30 minutes, centrifuged, and the supernatant was removed to obtain a Kluyveromyces yeast suspension. The Kluyveromyces yeast suspension was then subjected to the following post-treatment. The bacterial suspension was centrifuged, soaked in deionized water (5x volume) for 60 minutes, adjusted to pH 5.0 with food-grade lactic acid, and incubated at 50°C for 6 hours with 0.02% acid protease and 0.015% papain. The bacterial suspension was then incubated at 50°C for 6 hours, and particle size was measured using a laser particle size analyzer. The pH was adjusted to 6.6-6.8 with food-grade sodium bicarbonate, and 0.015% β-glucanase and 0.015% alkaline protease were added to the bacterial suspension. The bacterial suspension was incubated at 55°C for 24 hours, and particle size was measured using a laser particle size analyzer. The inactivated Kluyveromyces yeast cell suspension was decolorized by activated carbon adsorption and frozen at -20°C for 1 hour, cooled, and sterilized by immersion in 85% ethanol (3x volume) for 60 minutes. After recovering the ethanol, the bacterial suspension was mixed with deionized water in an amount equal to one weight of the suspension. Ozone was introduced into the solution for 1 hour for sterilization, and the solution was then pasteurized or autoclaved, followed by spray drying to obtain a dry powder of inactivated Kluyveromyces wickerhamii cells.

[0074] Example 4: Preparation of inactivated Kluyveromyces thermotolerans cell dry powder Kluyveromyces thermotolerans was cultured in a medium containing carbon sources, nitrogen sources, and salts (3.5% glucose, 0.7% molasses, 0.3% dry corn syrup, 0.02% magnesium sulfate, 0.6% ammonium sulfate, 0.7% yeast extract, 0.4% potassium dihydrogen phosphate, 1 ppm copper sulfate, 3 ppm ferrous sulfate, 15 ppm manganese sulfate, 3 ppm cobalt chloride, 10 ppm zinc sulfate, 15 ppm methionine, 3 ppm alanine, 1 ppm cysteine, and 9 ppm glycine). The pH of the medium was adjusted to 7.5-8.0, and the mixture was fermented at 30-35°C for 30 hours. The mixture was then heat-inactivated at 100°C for 25 minutes, centrifuged, and the supernatant was removed to obtain a Kluyveromyces yeast suspension. The Kluyveromyces yeast suspension was then subjected to the following post-treatment. The bacterial suspension was centrifuged, soaked in deionized water (5x volume) for 30 minutes, adjusted to pH 5.0 with food-grade lactic acid, and incubated at 50°C for 12 hours with 0.01% acid protease and 0.01% papain. The bacterial suspension was then incubated at 50°C for 12 hours and the particle size was measured using a laser particle size analyzer. The pH was adjusted to 6.6-6.8 with food-grade sodium bicarbonate, and the bacterial suspension was incubated at 55°C for 30 hours with 0.02% β-glucanase and 0.01% alkaline protease. The bacterial suspension was then decolorized by activated carbon adsorption to obtain an inactivated Kluyveromyces yeast cell suspension. This suspension was then frozen at -20°C for 1 hour, cooled, and sterilized by immersion in 85% ethanol (3x volume) for 60 minutes. After recovering the ethanol, the bacterial suspension was mixed with deionized water in an amount equal to its weight. Ozone was introduced into the solution for 1 hour for sterilization, and the solution was then pasteurized or autoclaved, followed by spray drying to obtain a dry powder of inactivated Kluyveromyces thermotolerans cells.

[0075] Example 5: Preparation of inactivated Kluyveromyces marxianus cell dry powder Kluyveromyces marxianus was cultured in a medium containing carbon and nitrogen sources and salts (4.5% glucose, 0.2% molasses, 0.2% dry corn syrup, 0.05% magnesium sulfate, 0.5% ammonium sulfate, 0.8% yeast extract, 0.6% potassium dihydrogen phosphate, 10 ppm copper sulfate, 5 ppm ferrous sulfate, 15 ppm manganese sulfate, 7 ppm cobalt chloride, 2 ppm zinc sulfate, 3 ppm methionine, 1 ppm alanine, 6 ppm cysteine, and 3 ppm glycine). The pH of the medium was adjusted to 5.5-6.5, and the mixture was fermented at 25-30°C for 24 hours. The mixture was then heat-inactivated at 100°C for 30 minutes, centrifuged, and the supernatant was removed to obtain a Kluyveromyces yeast suspension. The Kluyveromyces yeast suspension was then subjected to the following post-treatment. The bacterial suspension was centrifuged, soaked in deionized water (5x volume) for 30 minutes, adjusted to pH 5.0 with food-grade lactic acid, and incubated at 50°C for 6 hours with 0.015% acid protease and 0.015% papain. The slurry was then analyzed using a laser particle size analyzer. The pH was adjusted to 6.6-6.8 with food-grade sodium bicarbonate, and incubated at 55°C for 24 hours with 0.01% β-glucanase and 0.02% alkaline protease. The yeast was then decolorized by activated carbon adsorption to obtain an inactivated Kluyveromyces yeast suspension. This suspension was then frozen at -20°C for 1 hour, cooled, and sterilized by immersion in 85% ethanol (3x volume) for 60 minutes. After recovering the ethanol, the bacterial suspension was mixed with deionized water in an amount twice its weight. Ozone was introduced into the solution for 1 hour for sterilization, and the solution was then pasteurized or autoclaved, followed by spray drying to obtain a dry powder of inactivated Kluyveromyces marxianus cells.

[0076] Example 6: Preparation of inactivated Kluyveromyces lactis cell dry powder Kluyveromyces lactis was cultured in a medium containing carbon, nitrogen, and salts (4.2% glucose, 0.5% molasses, 0.1% dry corn syrup, 0.15% magnesium sulfate, 0.6% ammonium sulfate, 0.9% yeast extract, 0.5% potassium dihydrogen phosphate, 5 ppm copper sulfate, 10 ppm ferrous sulfate, 10 ppm manganese sulfate, 5 ppm cobalt chloride, 18 ppm zinc sulfate, 5 ppm methionine, 6 ppm alanine, 3 ppm cysteine, and 1 ppm glycine). The pH of the medium was adjusted to 4.0-5.0, and the mixture was fermented at 40-45°C for 28 hours. The mixture was then heat-inactivated at 100°C for 20 minutes, centrifuged, and the supernatant was removed to obtain a Kluyveromyces yeast suspension. The Kluyveromyces yeast suspension was then subjected to the following post-treatment. The bacterial suspension was centrifuged, soaked in deionized water (5x volume) for 60 minutes, adjusted to pH 5.0 with food-grade lactic acid, and incubated at 50°C for 6 hours with 0.02% acid protease and 0.02% papain. The slurry was then analyzed using a laser particle size analyzer. The pH was adjusted to 6.6-6.8 with food-grade sodium bicarbonate, and incubated at 55°C for 36 hours with 0.01% β-glucanase and 0.015% alkaline protease. The slurry was then decolorized by activated carbon adsorption to obtain an inactivated Kluyveromyces yeast cell suspension. This suspension was then frozen at -20°C for 1 hour, cooled, and sterilized by immersion in 85% ethanol (3x volume) for 60 minutes. After recovering the ethanol, the bacterial suspension was mixed with deionized water in an amount equal to the weight of the bacterial suspension. Ozone was introduced into the solution for 1 hour for sterilization, and the solution was then pasteurized or autoclaved, followed by spray drying to obtain a dry powder of inactivated Kluyveromyces lactis cells.

[0077] Example 7: Measurement of nutrients in the dried powders of Kluyveromyces yeast cells prepared in Examples 2 to 6 The dried powders of Kluyveromyces yeast cells prepared in Examples 2 to 6 were analyzed for protein, fat, and dietary fiber content, as well as moisture, ash, and total nitrogen. Moisture content was measured by the atmospheric dry weight method (105°C, 3 hours), total nitrogen by the Kjeldahl method, ash by the direct ashing method in a muffle furnace, dietary fiber content according to the AOAC 991.43 standard, protein content according to the AOAC 979.09 standard, fat content according to the AOAC 996.06 standard, moisture content according to the AOAC 925.09 standard, and ash content according to the AOAC 942.05 standard. The analytical results are shown in Table 2.

[0078] Table 2: Nutrients of the dried powder of Kluyveromyces yeast cells prepared in Examples 2 to 6 [Table 2]

[0079] Example 8: Experiments on composition stabilization systems The inactivated Kluyveromyces yeast cell dry powder prepared in Example 5 can be used as a food ingredient in the development of food formulations to replace part or all of the emulsifiers, thickeners, and stabilizers in foods.

[0080] Figures 6a-h show the stability of inactivated Kluyveromyces marxianus (KM) solutions in two different systems (oil-in-water emulsions and water-in-oil emulsions) and four different formulations (pure water (Figure 6a), oil-in-water (Figure 6b-d), pure oil (Figure 6e), and water-in-oil (Figure 6f-h)), as observed under a 400x microscope. Figures 6i and 6j show photographs of the composition systems at two different formulation ratios and conditions. Figure 6i shows an example of a composition system that showed no stratification or demulsification after 7 days but showed water-oil stratification after 14 days, while Figure 6j shows a stabilized composition system that showed no stratification or demulsification after 14 days.

[0081] Food compositions containing inactivated Kluyveromyces yeast cells are stable and homogeneous (no stratification or demulsification) without the use of exogenously added emulsifiers, thickeners, or stabilizers. Specifically, they were analyzed according to three characteristics (defined as A: no stratification or demulsification at all, B: slight demulsification with a small amount of stratification on top of the product, and C: obvious demulsification with complete separation of oil and water). 1. When the ratio of water to oil in a composition system is greater than that of oil, an oil-in-water emulsion is formed. The particle diameter of the inactivated, undisrupted Kluyveromyces cells in the aqueous phase is in the range of 2-5 μm, causing them to aggregate and act as a "caulking" agent. After emulsification of this composition system, the oil particles are in the range of 1-100 μm in diameter. The caulking effect of the smaller inactivated, undisrupted Kluyveromyces yeast cells physically isolates the oil particles from contact with each other, thereby delaying the time for emulsion breakdown. 2. In the composition system, as the proportion of oil increases, a water-in-oil system is formed. In this system, the oil particle size increases, and due to polarity, the inactivated, undisrupted Kluyveromyces cells in the oil particles tend to aggregate in the aqueous phase, ultimately forming fine particles with a particle size range of 10 to 100 μm in the aqueous phase. These fine particles physically isolate the oil particles from each other and delay the time for emulsification. 3. In the composition system, no water was added and the mixture consisted of pure olive oil and inactivated, undisrupted Kluyveromyces cells. The inactivated Kluyveromyces cells dispersed in the oil system, forming a solution system with a central cell cluster with a particle diameter of 50–100 μm and an outer layer of oil. However, this solution system underwent emulsion breakdown and stratification after 7 days, indicating the composition system's low stability (Table 3, Group 5, and Figure 6, e).

[0082] Based on a method for finding an appropriate water-oil ratio based on a constant yeast loading, it is estimated that a theoretical steady state will be reached when the compositions described herein contain the following amounts of inactivated Kluyveromyces yeast cells, oil, and water: Inactivated Kluyveromyces yeast cells 10-70% Edible oil 10~40% Water remaining

[0083] Table 3 Experimental composition ratio [Table 3]

[0084] Example 9: Preparation of a beverage containing inactivated Kluyveromyces yeast cells and rapeseed oil The inactivated Kluyveromyces yeast cell dry powder obtained in Examples 2 to 6 was emulsified with rapeseed oil and water at a certain weight percentage to prepare food and beverages with a desired particle size distribution. The specific operation steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was mixed with water and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, rapeseed oil was slowly added during the shearing process, and emulsification was continued while samples were taken every 2 minutes and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was further continued until the desired particle size distribution was achieved, and then the process was stopped to obtain a food composition beverage containing inactivated Kluyveromyces yeast cells. The stability of the product was observed at 25°C.

[0085] The weight percent contents, particle size distribution, and stability test results of the inactivated Kluyveromyces yeast cell dry powder, rapeseed oil, and water are shown in Tables 4, 5, 6, 7, and 8 below (A means no stratification or demulsification phenomena, B means slight demulsification with a small amount of stratification on top of the product, and C means obvious demulsification with the product showing complete separation of oil and water).

[0086] Table 4. Experimental results of the composition, particle size distribution, and stability of the composition in the beverage containing the inactivated Kluyveromyces yeast cells of Example 2 [Table 4-1] [Table 4-2]

[0087] Table 5. Experimental results of the composition, particle size distribution, and stability of the composition in the beverage containing the inactivated Kluyveromyces yeast cells of Example 3. [Table 5-1] [Table 5-2]

[0088] Table 6. Experimental results of composition, particle size distribution and stability of the composition in the beverage containing the inactivated Kluyveromyces yeast cells of Example 4 [Table 6-1] [Table 6-2]

[0089] Table 7. Experimental results of the composition, particle size distribution, and stability of the composition in the beverage containing the inactivated Kluyveromyces yeast cells of Example 5. [Table 7-1] [Table 7-2]

[0090] Table 8. Experimental results of composition, particle size distribution and stability of the composition in the beverage containing the inactivated Kluyveromyces yeast cells of Example 6. [Table 8-1] [Table 8-2]

[0091] As shown in Tables 4 to 8, when the ratio of inactivated Kluyveromyces yeast cell powder was 2.5 to 25%, the ratio of edible oil was 1% to 40%, and the ratio of water was 40% to 96.5%, shear emulsification was performed to reduce the particle size of each component in the suspension to 100 μm or less (100% frequency of particle sizes ≦100 μm). A stable and uniform state was maintained at room temperature for 7 days without stratification. When the ratio of inactivated Kluyveromyces yeast cell powder was 2.5 to 25% and the ratio of edible oil was 1 to 30%, shear emulsification was similarly performed to reduce the particle size of each component to 100 μm or less. A stable and uniform state was maintained at room temperature for 28 days without stratification.

[0092] Example 10 Preparation of a sauce containing inactivated Kluyveromyces yeast and rapeseed oil The inactivated Kluyveromyces yeast cell dry powder obtained in Examples 2 to 6 was emulsified with rapeseed oil and water at a certain weight percentage to prepare a food sauce with a desired particle size distribution. The specific operation steps are as follows: (1) Kluyveromyces yeast cell dry powder was mixed with water and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, rapeseed oil was slowly added during the shearing process, and emulsification was continued while samples were taken every 2 minutes and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was continued until the desired particle size distribution was achieved, and then the emulsification was stopped to obtain a food composition sauce containing inactivated Kluyveromyces yeast cells, and the stability of the product was observed under conditions of 25°C.

[0093] The weight percent contents of the inactivated Kluyveromyces yeast cell dry powder, rapeseed oil, and water, the particle size of the compositions, and the stability test results are shown in Tables 9, 10, 11, 12, and 13, respectively (A indicates no stratification or demulsification, B indicates slight demulsification with a small amount of stratification on top of the product, and C indicates obvious demulsification with complete separation of oil and water).

[0094] Table 9. Experimental results of composition, particle size distribution and stability of the composition in the sauce containing inactivated Kluyveromyces yeast cells from Example 2. [Table 9-1] [Table 9-2]

[0095] Table 10. Experimental results of composition, particle size distribution, and stability of the composition in the sauce containing inactivated Kluyveromyces yeast cells of Example 3 [Table 10-1] [Table 10-2]

[0096] Table 11. Experimental results of composition, particle size distribution, and stability of the composition in the sauce containing inactivated Kluyveromyces yeast cells from Example 4 [Table 11-1] [Table 11-2]

[0097] Table 12. Experimental results of composition, particle size distribution, and stability of the composition in the sauce containing inactivated Kluyveromyces yeast cells of Example 5 [Table 12-1] [Table 12-2]

[0098] Table 13. Experimental results of composition, particle size distribution and stability of the composition in the sauce containing inactivated Kluyveromyces yeast cells of Example 6 [Table 13-1] [Table 13-2]

[0099] As shown in Tables 9 to 13, when the ratio of inactivated Kluyveromyces yeast cell powder was 30 to 70%, the ratio of edible oil was 1 to 30%, and the ratio of water was 18 to 69%, shear emulsification was performed so that the particle size of each component in the suspension was 100 μm or less (the frequency of particle sizes ≦100 μm was 100%), and a stable and uniform state was maintained at room temperature for 28 days without stratification.

[0100] Example 11 Preparation of a beverage containing inactivated Kluyveromyces marxianus cells and coconut oil The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with coconut oil and water at a certain weight percentage to prepare a food beverage with a desired particle size distribution. The specific steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, coconut oil was slowly added during the shearing process, and emulsification was continued while samples were taken every 2 minutes and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was further continued until the desired particle size distribution was reached, and then the process was stopped to obtain a food composition beverage containing inactivated Kluyveromyces yeast cells. The stability of the product was observed at 25°C.

[0101] The weight percent contents of inactivated Kluyveromyces yeast cell dry powder, coconut oil, and water, particle size distribution of the compositions, and stability test results are also shown in Table 14 below (A is defined as no stratification or demulsification phenomena, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0102] Table 14. Experimental results of composition, particle size distribution and stability in beverages [Table 14-1] [Table 14-2]

[0103] Example 12 Preparation of a sauce containing inactivated Kluyveromyces marxianus cells and coconut oil A food source with a desired particle size distribution was prepared by emulsifying the inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 with coconut oil and water at a certain weight percent content. The specific steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, coconut oil was slowly added during the shearing process, and emulsification was maintained. Samples were taken every 2 minutes, and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was continued until the desired particle size distribution was achieved, and then the emulsification was stopped to obtain a source of a food composition containing inactivated Kluyveromyces yeast cells. The stability of the product was observed at 25°C.

[0104] The weight percent contents of inactivated Kluyveromyces yeast cell dry powder, coconut oil, and water, particle size distribution of the compositions, and stability experimental results are shown in Table 15 below (A is defined as no stratification or demulsification phenomena, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0105] Table 15. Experimental results of composition, particle size distribution and stability in sauce [Table 15-1] [Table 15-2]

[0106] Example 13 Preparation of a beverage containing inactivated Kluyveromyces marxianus cells and high oleic sunflower oil The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with high oleic acid sunflower oil and water at a certain weight percent content to prepare a food beverage with a desired particle size distribution. The specific operation steps are as follows: (1) Add the dried powder of Kluyveromyces yeast cells to water to adjust the volume to the corresponding concentration and stir until uniformly dispersed; (2) Shear emulsification was initiated using an emulsifier, and high oleic acid sunflower oil was slowly added during the shearing process to maintain emulsification. Samples were taken every 2 minutes, and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was then stopped to obtain a food composition beverage containing inactivated Kluyveromyces yeast cells, and the stability of the product was observed at 25°C.

[0107] The weight percent contents of the inactivated Kluyveromyces yeast cell dry powder, high oleic sunflower oil, and water, the particle size distribution of the compositions, and the experimental results of the stability are also shown in Table 16 below (A is defined as no stratification or demulsification phenomenon at all, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0108] Table 16. Experimental results of composition, particle size distribution and stability in beverages [Table 16-1] [Table 16-2]

[0109] Example 14 Preparation of a sauce containing inactivated Kluyveromyces marxianus cells and high oleic sunflower oil The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with high oleic acid sunflower oil and water at a certain weight percent content to prepare a food sauce with a desired particle size distribution. The specific operation steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, and high oleic acid sunflower oil was slowly added during the shearing process to maintain emulsification. Samples were taken every 2 minutes, and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was continued until the desired particle size distribution was achieved, and then the process was stopped to obtain a food composition sauce containing inactivated Kluyveromyces yeast cells. The stability of the product was observed at 25°C.

[0110] The weight percent contents of the inactivated Kluyveromyces yeast cell dry powder, high oleic sunflower oil, and water, the particle size distribution of the compositions, and the stability test results are also shown in Table 17 below (A is defined as no stratification or demulsification phenomena, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0111] Table 17. Experimental results of composition, particle size distribution and stability in sauce [Table 17-1] [Table 17-2]

[0112] Example 15 Preparation of a beverage containing inactivated Kluyveromyces marxianus cells and canola oil The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with canola oil and water at a certain weight percentage to prepare a food beverage with a desired particle size distribution. The specific operation steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, canola oil was slowly added during the shearing process, and emulsification was continued while samples were taken every 2 minutes and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was further continued until the desired particle size distribution was achieved, and then the emulsification was stopped to obtain a food composition beverage containing inactivated Kluyveromyces yeast cells, and the stability of the product was observed under conditions of 25°C.

[0113] The weight percent contents of inactivated Kluyveromyces yeast cell dry powder, canola oil, and water, the particle size distribution of the compositions, and the stability test results are also shown in Table 18 below (A is defined as no stratification or demulsification, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0114] Table 18. Experimental results of composition, particle size distribution and stability of the components in the beverage [Table 18-1] [Table 18-2]

[0115] Example 16 Preparation of a sauce containing inactivated Kluyveromyces marxianus cells and canola oil The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with canola oil and water at a certain weight percentage content to prepare a food source with a desired particle size distribution. The specific operation steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, canola oil was slowly added during the shearing process, and emulsification was continued while samples were taken every 2 minutes and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was further continued until the desired particle size distribution was achieved, and then the emulsification was stopped to obtain a food composition source containing inactivated Kluyveromyces yeast cells, and the stability of the product was observed under conditions of 25°C.

[0116] The weight percent contents of inactivated Kluyveromyces yeast cell dry powder, canola oil, and water, the particle size distribution of the compositions, and the stability test results are also shown in Table 19 below (A is defined as no stratification or demulsification, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0117] Table 19. Experimental results of composition, particle size distribution and stability of ingredients in sauce [Table 19-1] [Table 19-2]

[0118] Example 17 Preparation of a Beverage Containing Inactivated Kluyveromyces marxianus Cells and Medium-Chain Triglycerides The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with medium-chain triglycerides and water at a certain weight percent content to prepare a food beverage with a desired particle size distribution. The specific operation steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, and a medium-chain triglyceride was slowly added during the shearing process to maintain emulsification. Samples were taken every 2 minutes, and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was further continued until the desired particle size distribution was achieved, and then the process was stopped. A food composition beverage containing inactivated Kluyveromyces yeast cells was obtained, and the stability of the product was observed at 25°C.

[0119] Among them, the weight percent contents of inactivated Kluyveromyces yeast cell dry powder, medium-chain triglyceride, and water, the particle size distribution of the composition, and the stability test results are shown in Table 20 (A means no stratification or demulsification phenomenon at all, B means slight demulsification with a small amount of stratification on the top of the product, and C means obvious demulsification with complete separation of oil and water in the product).

[0120] Table 20. Experimental results of composition, particle size distribution and stability of components in beverages [Table 20-1] [Table 20-2]

[0121] Example 18 Preparation of a Source Containing Inactivated Kluyveromyces marxianus Cells and Medium-Chain Triglycerides The inactivated Kluyveromyces yeast cell dry powder obtained in Example 5 was emulsified with medium-chain triglycerides and water at a certain weight percent content to prepare a food sauce with a desired particle size distribution. The specific operation steps are as follows: (1) Dry powder of Kluyveromyces yeast cells was added to water to the corresponding concentration and stirred until uniformly dispersed. (2) Shear emulsification was initiated using an emulsifier, and a medium-chain triglyceride was slowly added during shearing to maintain emulsification. Samples were taken every 2 minutes, and the particle size distribution of the samples was detected using a laser particle size analyzer. The emulsification was then stopped to obtain a food composition sauce containing inactivated Kluyveromyces yeast cells, and the stability of the product was observed under conditions of 25°C.

[0122] The weight percent contents of inactivated Kluyveromyces yeast cell dry powder, medium-chain triglycerides, and water, the particle size distribution of the compositions, and the stability test results are shown in Table 21 (A is defined as no stratification or demulsification phenomena, B is defined as slight demulsification with a small amount of stratification on top of the product, and C is defined as obvious demulsification with the product showing complete separation of oil and water).

[0123] Table 21 Experimental results of composition, particle size distribution and stability of ingredients in sauce [Table 21-1] [Table 21-2]

[0124] The experimental results in Tables 7, 14, 16, 18, and 20 confirm that when inactivated Kluyveromyces marxianus cells are added to rapeseed oil, coconut oil, high oleic sunflower oil, canola oil, medium-chain triglycerides, or the like in the proportions described herein to prepare the food and beverages described herein, and emulsified by shear until the particle size distribution of each component in the suspension is 100 μm or less (100% particle size distribution of ≦100 μm), the food and beverages can maintain a stable, homogeneous, and non-stratified state at room temperature for at least 7 days. In the case of the preferred proportions of the beverages described herein (2.5-25% inactivated Kluyveromyces yeast cell powder and 1%-30% edible oil), the food and beverages can even maintain a stable, homogeneous, and non-stratified state for 28 days.

[0125] The experimental results in Tables 9, 15, 17, 19, and 21 confirm that when the food sauce described herein is prepared by adding inactivated Kluyveromyces marxianus cells to rapeseed oil, coconut oil, high oleic sunflower oil, canola oil, medium-chain triglycerides, etc. in the blending ratios described herein, and emulsifying by shear until the particle size distribution of each component in the suspension is 100 μm or less (100% particle size frequency of 100 μm or less), a stable, uniform, non-stratified state can be maintained at room temperature for at least 28 days.

[0126] In particular, the above-mentioned edible oils are all commonly used in the food industry with different sources and nutritional compositions, and their commercial prices range from 10 to 300 RMB / L (canola oil 10 RMB / L, high oleic sunflower oil 16 RMB / L, coconut oil 50 RMB / L, medium-chain triglycerides 300 RMB / L), and the food compositions described in the present disclosure are inexpensively available in terms of raw materials and relatively low in terms of cost.

[0127] It should be understood that Examples 11 to 18 are illustrative examples, and that the other four inactivated Kluyveromyces yeast cells described in the present disclosure (Kluyveromyces hubeiensis, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, and Kluyveromyces lactis) other than inactivated Kluyveromyces marxianus cells have shelf life stability that is the same as or similar to that of inactivated Kluyveromyces marxianus cells when used in food, beverages, or sauces prepared with edible oils other than rapeseed oil in the blending ratios described in the present disclosure.

[0128] Example 19: Stability study of foods prepared with food compositions containing inactivated Kluyveromyces marxianus cells In Example 5, a Kluyveromyces beverage was prepared by shear emulsification of inactivated Kluyveromyces marxianus cell dry powder. This food composition was replaced with pea protein and sucrose fatty acid esters in the same manner and proportions to obtain a plant-based beverage and an emulsifier beverage. The compositions of the beverage compositions are shown in Table 22. Furthermore, stratification and demulsification of the three beverages were recorded on days 1, 3, 7, 14, and 28 after preparation (A: no stratification or demulsification at all; B: slight demulsification with a small amount of stratification on top of the product; C: obvious demulsification with complete separation of oil and water). The observed results are shown in Table 23.

[0129] Table 22. Composition of beverage composition [Table 22]

[0130] Table 23. Stability experiment results [Table 23]

[0131] As shown in Table 23, the Kluyveromyces beverage formulated without exogenous food additives maintained a stable, uniform suspension without demulsification or stratification throughout the 28-day shelf-life observation period. The plant beverage formulated without exogenous food additives exhibited significant stratification and demulsification on Day 7 of the 28-day shelf-life observation period. The emulsified beverage containing the emulsifying food additive sucrose fatty acid ester exhibited slight stratification and demulsification on Day 7 of the 28-day shelf-life observation period, but exhibited significant stratification and demulsification on Day 28. These findings demonstrate that Kluyveromyces beverages are valuable for the development of microbial beverages and microbial food ingredients.

[0132] The foregoing descriptions of specific exemplary embodiments of the present disclosure have been given for purposes of explanation and illustration. These descriptions are not intended to limit the disclosure to the precise forms disclosed, and obviously, many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the present disclosure and their practical application, thereby enabling those skilled in the art to realize and utilize various different exemplary embodiments of the present disclosure, as well as various different options and modifications. It is intended that the scope of the present disclosure be limited by the claims and their equivalents. <Additional Notes> The present invention includes the following aspects. <Section 1> A composition comprising, as raw materials, 2.5 to 80% by weight of inactivated Kluyveromyces yeast cells, 1 to 50% by weight of an edible oil, and 18 to 96.5% by weight of water. <Section 2> The composition according to <Item 1>, wherein the particle size of the inactivated yeast cells of the genus Kluyveromyces is 1 to 7 μm, and preferably the particle size of the inactivated yeast cells of the genus Kluyveromyces is 2 to 5 μm. <Section 3> The composition according to item 1 or 2, wherein the inactivated Kluyveromyces yeast cells contain 30 to 55% protein, 1 to 5% fat, and 15 to 30% dietary fiber. <Section 4> the inactivated Kluyveromyces yeast cells are one or more selected from Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickerhamii, and Kluyveromyces thermotolerans cells; Preferably, the composition according to any one of the above <Items>, wherein the inactivated Kluyveromyces yeast cells are Kluyveromyces marxianus and / or Kluyveromyces lactis cells. <Section 5> The composition according to any one of the above items 1, wherein the particle size of the edible oil is 1 to 100 μm. <Section 6> the inactivated Kluyveromyces yeast cells are dried inactivated Kluyveromyces yeast cells, dehydrated inactivated Kluyveromyces yeast cells, or a suspension of inactivated Kluyveromyces yeast cells; Preferably, the inactivated Kluyveromyces yeast cells are dried inactivated Kluyveromyces yeast cells or an inactivated Kluyveromyces yeast cell suspension; Preferably, the edible oil is one or more selected from the group consisting of soybean oil, rapeseed oil, high oleic acid sunflower oil, medium chain triglycerides, canola oil, coconut oil, corn oil, sesame oil, tea seed oil, rice bran oil, olive oil, linseed oil, safflower oil, grapeseed oil, walnut oil, palm oil, peanut oil, and blended oils. <Section 7> The composition according to any one of <Item 1>, wherein the inactivated Kluyveromyces yeast cells are prepared by a method comprising the steps of selecting a medium containing a carbon source, a nitrogen source, and salts, and culturing a Kluyveromyces yeast strain for 15 to 40 hours; and adjusting the pH of the medium to 4.0 to 8.0, fermenting the medium at 25 to 50°C, and then heat-inactivating the medium to obtain the inactivated Kluyveromyces yeast cells. <Section 8> A method for preparing a composition, comprising the steps of: mixing inactivated Kluyveromyces yeast cells with water to form a cell suspension; and adding an edible oil during shear emulsification of the cell suspension, and continuously shearing and emulsifying the edible oil until the particle size of the edible oil reaches 1 to 100 μm. <Section 9> A food product comprising the composition according to any one of <Item 1> to <Item 7>, Preferably, when the food product is a beverage, the composition comprises, as ingredients, 2.5 to 25% by weight of inactivated Kluyveromyces yeast cells, 1 to 40% by weight of an edible oil, and 40 to 96.5% by weight of water; Preferably, when the food is a sauce, the composition comprises, as ingredients, 30 to 70% by weight of inactivated Kluyveromyces yeast cells, 1 to 30% by weight of edible oil, and 18 to 69% by weight of water. <Section 10> Use of the composition according to any one of <Item 1> to <Item 7> in the preparation of food, Preferably for use in the preparation of biscuits, bread, baked goods, puffed goods, freeze-dried goods, ice cream, dehydrated and dried goods.

Claims

1. A composition comprising, as raw materials, 2.5 to 70% by weight of inactivated Kluyveromyces yeast cells, 1 to 40% by weight of edible oil, and 18 to 96.5% by weight of water, wherein the edible oil is dispersed in the form of droplets having a particle size of 1 to 100 μm in an aqueous phase containing the inactivated Kluyveromyces yeast cells, the inactivated Kluyveromyces yeast cells are Kluyveromyces marxianus and / or Kluyveromyces lactis cells, and the particle size of the inactivated Kluyveromyces yeast cells is 2 to 7 μm.

2. 2. The composition of claim 1, wherein the inactivated Kluyveromyces yeast cells comprise 30-55% protein, 1-5% fat, and 15-30% dietary fiber.

3. 2. The composition of claim 1, wherein the inactivated Kluyveromyces yeast cells are dried inactivated Kluyveromyces yeast cells, dehydrated inactivated Kluyveromyces yeast cells, or a suspension of inactivated Kluyveromyces yeast cells.

4. The composition described in claim 1, characterized in that the inactivated Kluyveromyces yeast cells are dried inactivated Kluyveromyces yeast cells or an inactivated Kluyveromyces yeast cell suspension.

5. The composition of claim 1, characterized in that the edible oil is one or more selected from soybean oil, rapeseed oil, high oleic sunflower oil, medium chain triglyceride, canola oil, coconut oil, corn oil, sesame oil, tea seed oil, rice bran oil, olive oil, linseed oil, safflower oil, grapeseed oil, walnut oil, palm oil, peanut oil, and blended oils.

6. A method for preparing the inactivated Kluyveromyces yeast cells, comprising: The preparation method includes the steps of selecting a medium containing a carbon source, a nitrogen source, and salts, and culturing a yeast strain of the genus Kluyveromyces for 15 to 40 hours; adjusting the pH of the medium to 4.0 to 8.0, fermenting the medium at 25 to 50°C, and then heat-inactivating the medium to obtain the inactivated yeast cells of the genus Kluyveromyces; 2. The method of claim 1, wherein the inactivated Kluyveromyces yeast cells are Kluyveromyces marxianus and / or Kluyveromyces lactis cells and are contained in the composition of claim 1.

7. A method for preparing the composition of claim 1, comprising the steps of: mixing inactivated Kluyveromyces yeast cells with water to form a cell suspension; and adding edible oil during shear emulsification of the cell suspension, and continuously shear emulsifying the edible oil until the particle size of the edible oil reaches 1 to 100 μm.

8. A food product comprising the composition of claim 1.

9. The food described in claim 8, wherein when the food is a beverage, the composition comprises, as ingredients, 2.5 to 25% by weight of inactivated Kluyveromyces yeast cells, 1 to 40% by weight of edible oil, and 40 to 96.5% by weight of water.

10. The food of claim 8, wherein when the food is a sauce, the composition comprises, as ingredients, 30 to 70% by weight of inactivated Kluyveromyces yeast cells, 1 to 30% by weight of edible oil, and 18 to 69% by weight of water.

11. 10. Use of the composition of claim 1 in the preparation of food products.

12. The use described in claim 11, wherein the use is the preparation of biscuits, bread, baked foods, puffed foods, freeze-dried foods, ice cream, and dehydrated dried foods.

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