Method for increasing probiotic viability in food products
阅读说明：本技术 增加食品中益生菌活力的方法 (Method for increasing probiotic viability in food products ) 是由 泽纳布·阿里 瓦瑞德哈瑞詹·瑞德哈马尼·巴丝克 陈予敏 考杜拉·弗兰克龙 胡安·冈萨雷斯 于 2019-06-10 设计创作，主要内容包括：预调理的益生菌组合物和形成预调理的益生菌组合物的方法包括将解冻的益生菌细胞引入预调理环境中,所述预调理环境具有足够的营养物和条件以制备用于在目标食品中存活并成功繁殖的解冻的益生菌细胞。然后为解冻的益生菌细胞提供至少2小时的孵育期以产生接种预处理的益生菌细胞,所述预处理的益生菌细胞在食品中具有改善的货架寿命。(A preconditioned probiotic composition and method of forming a preconditioned probiotic composition include introducing thawed probiotic cells into a preconditioned environment with sufficient nutrients and conditions to prepare the thawed probiotic cells for survival and successful propagation in a target food product. The thawed probiotic cells are then provided with an incubation period of at least 2 hours to produce inoculated pretreated probiotic cells having improved shelf life in a food product.)
1. A method of forming stable probiotic cells, comprising:
introducing thawed probiotic cells into a preconditioned environment comprising sufficient nutrients to prepare said thawed probiotic cells for incorporation into a food product; and
preconditioning the thawed probiotic cells in the preconditioning environment for at least about 2 hours, thereby producing preconditioned probiotic cells.
2. The method of claim 1, wherein the preconditioned environment comprises apple juice.
3. The method of claim 1, wherein the preconditioned environment comprises a fluid, and the fluid and probiotic cells are blended to achieve a predetermined probiotic concentration.
4. The method of claim 3, wherein the predetermined probiotic concentration is about 1.0 x 1010CFU/mL to 1.0X 1011CFU/mL。
5. The method of claim 1, wherein the preconditioned environment comprises a temperature of from about 0 ℃ to about 39 ℃.
6. The method of claim 1, wherein the preconditioned environment comprises a temperature of from about 0 ℃ to about 10 ℃.
7. The method of claim 1, wherein the incubation period is less than one complete cell division cycle.
8. The method of claim 7, wherein the at least one complete cell division cycle is less than 24 hours.
9. The method of claim 1, comprising thawing the frozen probiotic cells prior to said introducing step.
10. The method of claim 9, comprising thawing the frozen probiotic cells at a temperature of about 0 ℃ to 39 ℃.
11. The method according to claim 9, characterized in that it comprises thawing the frozen probiotic cells for at least 8 hours.
12. The method according to claim 1, comprising, after the providing step, adding the pre-treated probiotic cells to a pasteurized product matrix to produce a food product comprising stabilized probiotic cells. The method of claim 1, wherein the nutrient comprises a carbohydrate.
13. The method of claim 1, wherein the nutrients comprise amino acids.
14. The method of claim 14, wherein the amino acid comprises homocysteine, cystathionine, cysteine, or a combination thereof.
15. A probiotic blend for incorporation into a pasteurized product matrix to provide a preconditioning of a food product, comprising:
a predetermined amount of thawed probiotic cells;
a preconditioning fluid, wherein the preconditioning fluid comprises a nutrient; and
about 1.0X 1010CFU/mL to about 1.0X 1011Probiotic concentration of CFU/mL.
16. The probiotic blend according to claim 16, characterized in that the pasteurized product matrix has a pH below about 7.0.
17. The probiotic blend according to claim 16, characterized in that the pasteurized product matrix has a first pH and the preconditioned probiotic blend has a second pH, wherein the first pH differs from the second pH by 1.0pH or less.
18. The probiotic blend according to claim 16, characterized in that the food product is a beverage.
19. The probiotic blend according to claim 16, wherein at least 10 hundred million CFU of probiotic cells are provided per 8 ounce (236mL) serving of the food product.
Technical Field
The present invention relates to food products comprising probiotic organisms and to a method of producing robust probiotic cells to be incorporated into a variety of food products.
Background
In food products manufactured to incorporate live probiotic bacteria, the live probiotic cells in certain food products are typically substantially reduced (e.g., by one log or more) at the end of the expected shelf life of the product. One current solution to this reduction involves incorporating an excess of probiotics into the product when producing a food product incorporating the probiotics to maximize the amount of viable probiotic bacteria remaining over the shelf life of the food product. There remains a need for more robust probiotic cells or methods of making probiotic cells robust in a variety of food products so that excess of probiotic can be avoided to the greatest extent.
Disclosure of Invention
The following is a simplified summary of the disclosure and is intended to provide a basic understanding of the methods and products described herein. It is not an exhaustive overview nor is it intended to identify key or critical elements or to delineate the scope of this specification. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.
An exemplary method of producing robust probiotic cells comprises the steps of: introducing the thawed probiotic cells into a preconditioned environment comprising sufficient nutrients to prepare the thawed probiotic cells for incorporation into a target food product; and providing the thawed probiotic cells with an incubation period of at least 2 hours, thereby producing inoculated pretreated probiotic cells. In some embodiments, the incubation period comprises up to 1 complete cycle of probiotic cell division. In some embodiments, the method comprises the step of thawing the frozen probiotic cells prior to the introducing step. In some embodiments, the method further comprises the step of adding the pretreated probiotic cells to a target food product after an incubation period to produce a food product comprising a robust probiotic supplement. Optionally, the treatment and preconditioning steps are performed simultaneously with the production of the target food product (i.e., food or beverage product) to which the probiotic bacteria are added. The inoculated probiotic cells may be incorporated with any number of fruits, vegetables and/or dairy products. In some embodiments, the food product comprises a low pH matrix, which may have a pH of less than about 4.5. Other aspects, embodiments, and features of the present invention will become apparent in the following written detailed description and the accompanying drawings.
Drawings
The novel features believed characteristic of the invention are set forth in the appended claims. The invention itself, however, as well as a preferred mode of use, further objectives and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying drawings.
FIG. 1 depicts a flow diagram of one embodiment of the methods described herein.
Fig. 2 depicts a schematic of the toxicity of a low pH food environment to probiotic cells and the detoxification mechanism of the cellular processes of the methods described herein.
Fig. 3 depicts a bar graph comparing the viability of probiotic cells in three samples in which probiotics were introduced into a beverage by direct inoculation without preconditioning, preconditioning at 4 ℃ for 2 hours, and preconditioning at 4 ℃ for 4 hours.
Detailed Description
Currently available probiotics are produced under conditions ideal for subsequent storage by freezing or freeze-drying prior to use. Such conditions are quite different from those of some food substrates into which probiotic bacteria are incorporated. For example, freshly thawed probiotic bacteria are not prepared to survive shelf life in the acidic conditions found in juice, such as orange juice, and include pH levels of about 4, aerobic environments, compounds that may be detrimental to probiotic viability, and nutrient levels that may not be enriched with amino acids or proteins suitable for probiotic survival. For example, phytochemicals (e.g., polyphenols) can adversely affect probiotic viability. Probiotic bacterial cells thawed from a frozen state may lack the structure and enzyme systems of the low pH environment that treats fruit-based beverages, including proton pumps on cell membranes (F0F 1-atpase) and cell buffer systems. Thus, the shelf life of the probiotic cells in the food product may be significantly reduced.
Under such widely varying beverage product conditions, the reduction in live probiotic bacterial cells is typically greater than about 95% over a period of 72 days after incorporation of the live probiotic bacterial cells into an acidic beverage (e.g., a fruit-based beverage). The underlying reason for the low survival rate of probiotics in fruit-based beverages is that probiotic bacterial cells are produced under conditions designed to maximize their yield and viability after storage. That is, the probiotic cells are designed to maximize recovery from damage from the freeze-thaw cycle. However, these conditions are not designed to allow the probiotic cells to adapt to survive in the product matrix of certain food products, including fruit-based beverages comprising low pH, and under the conditions of production of certain food products. There is no time and nutritional support for the probiotic cells to adapt to their intended/final food matrix. For example, fruit-based beverages containing small amounts of amino acids or proteins do not provide sufficient nutrition for probiotic bacteria to produce the large quantities of required proton pumps, cell buffer systems, and enzymes for detoxifying oxygen that enable cells to survive in harsh, low pH environments.
The bacterial growth cycle is divided into four phases: lag phase, log phase, stationary phase and death phase. During the lag phase, bacterial cells replicate various proteins and synthesize RNA in preparation for the log phase, during which the size of bacterial colonies increases dramatically. After log phase, stationary phase involves a slowing down of cell and colony growth. As resources are utilized, the rate of cell death begins to coincide with the rate of cell division. Colonies enter the final death phase when conditions decline due to limited resources, waste production, or other environmental changes.
By focusing on the conditions and physiology of the lag phase, it has been proposed to treat the probiotics to allow adaptation to certain growth conditions of the food substrate in which the probiotics are ultimately incorporated. By providing the bacteria with components that allow their culture medium to be stabilized in the proper balance, improved interaction with certain food substrates will result in improved shelf life of the probiotic. Fig. 1 is a flow diagram illustrating one embodiment of a method for increasing the probiotic viability in a pasteurized product matrix 0108.
In one embodiment, the treated probiotic cells are configured to survive at low pH by incorporating nutritional processing aids (e.g., carbohydrates and amino acids) and setting appropriate conditions for the probiotic cells to establish a cellular enzyme system. The method for forming stable or robust probiotic cells comprises providing frozen probiotic cells 0102. The frozen probiotic cells 0102 can be stored in a refrigerator at-80 ℃. In some embodiments, the method comprises thawing 0104 frozen probiotic cells. For example, the frozen probiotic cells 0102 may be placed in a storage environment at a temperature of 0 to 39 ℃. In some embodiments, the method comprises introducing thawed probiotic cells into a preconditioned environment 0106, the preconditioned environment 0106 containing sufficient nutrients to prepare the thawed probiotic cells for incorporation into the pasteurized food product; and preconditioning the thawed probiotic cells with an incubation period of at least 2 hours to produce inoculated pretreated probiotic cells. In some embodiments, the method may include thawing the frozen probiotic cells and then introducing the thawed probiotic cells into a preconditioned environment. In some embodiments, the method may include introducing thawed probiotic cells into the preconditioned environment while thawing the frozen probiotic cells. For example, the combined steps of thawing and introducing may be performed for about 2 hours to 24 hours. The conditions of the preconditioning environment may allow the probiotic cells to recover from freeze-thaw damage of the manufactured frozen probiotic cells. In some embodiments, the preconditioned probiotic cells are introduced into the pasteurized product matrix 0108 to provide a food product comprising probiotic cells. As used herein, a "pasteurized product base" can be in a semi-solid or liquid form (e.g., a snack or beverage). As used herein, a "food product" comprises a pasteurized product matrix and preconditioned probiotic cells. In some embodiments, the method includes packaging 0110 the food product in a sterile container.
In some embodiments, sufficient nutrients include one or more of carbohydrates and amino acids in order to obtain robust probiotic cells. In some embodiments, the nutrient comprises a carbohydrate, such as lactose, maltose, fructose, glucose, sucrose, and combinations thereof. In some embodiments, the nutrient includes other simple sugars or oligosaccharides naturally present in the juice. For example, juices, concentrates, purees, mannas (nectars) and other preparations may be derived from any one or combination of fruits such as apples, oranges, pears, mangos, pomegranates, watermelons, pineapples, peaches, grapes, kiwis, coconuts and lemons. In some embodiments, the nutrient includes a carbohydrate, for example, a juice or puree derived from vegetables such as wheatgrass, cucumber, lettuce, pumpkin, and white gourd. In some embodiments, the nutrient comprises one or more amino acids. In some embodiments, the amino acid comprises homocysteine. In some embodiments, the amino acid comprises cystathionine. In some embodiments, the amino acid comprises cysteine. In some embodiments, the amino acid comprises homocysteine, cystathionine, cysteine, or any combination or sub-combination thereof. Other suitable amino acids include, but are not limited to, alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, asparagine, cysteine, glutamine, methionine, serine, threonine, arginine, histidine, lysine, aspartic acid, glutamic acid, glycine, proline, or combinations or sub-combinations thereof. Without being bound by any particular theory, it is believed that the amino acids cause the probiotic cells to generate a proton pump and develop a cell buffer (cellular buffer) to cope with the low pH conditions of the food product to which the probiotics will be added to produce a food product with a robust probiotic supplement.
In addition to carbohydrates and amino acids, organic acids may also be present in a suitable preconditioning environment. Some examples of organic acids may include, but are not limited to, citric acid, malic acid, tartaric acid, succinic acid, formic acid, and lactic acid, or combinations or sub-combinations thereof.
In addition to sufficient balanced nutrients for successful propagation in the desired food matrix environment, the method includes providing the thawed probiotic cells with an incubation period of about 2 hours to a time taken for at most the probiotic cells to complete one cell division cycle. In some embodiments, the cell division cycle may comprise up to about 24 hours. In some embodiments, the cell division cycle can be about 6 hours to 16 hours. The cell division cycle time depends on many different factors including, for example, the environment of the probiotic cells, the quality of the preconditioning fluid, and the type of probiotic cells. The incubation period should allow and induce the probiotic cells to produce proton pumps and develop a cell buffer that is required for the food product, more specifically for the target food or the environment of the desired food product to which the pre-treated probiotic cells are to be added. Without wishing to be bound by any particular theory, it is believed that extending the incubation period beyond the cell division cycle may result in the organoleptic and nutritional values of the product deviating from the desired product specifications. For example, the process of cell division may involve further metabolism of nutrients in the environment, resulting in changes in the nutritional value and organoleptic characteristics of the product.
The treatment within the preconditioning environment may also include a suitable temperature to allow adaptation of the probiotic cells to the desired food matrix. In some embodiments, the preconditioning environment comprises a temperature of about 0 ℃ to 39 ℃. In some embodiments, the preconditioning environment comprises a temperature of about 4 ℃ to 10 ℃. In other embodiments, one or more temperatures may be selected to balance the energy cost of warming the cells and to optimize the time taken for thawing. In some embodiments, the time taken for thawing is about 1 to 16 hours. In some embodiments, the time taken for thawing is 8 to 16 hours. In some embodiments, the time taken for thawing is about 4 to 8 hours. The time taken for thawing can vary based on the temperature used to thaw the probiotic.
In some embodiments, the process 0101 shown in fig. 1 can be performed in parallel with the blending and pasteurization process of the fruit beverage or other food product. Fig. 2 depicts a schematic of the toxicity of the low pH environment of a fruit beverage to probiotic cells, the detoxification mechanism of a proton pump, and potentially critical cellular processes to treat the probiotic cells described herein. Outside the cells, the pH of the fruit beverage sample was 4. The low external pH promotes a large portion of the organic acids in a neutral form. The neutral form of this organic acid diffuses into the cell. Inside the cell, the high internal pH (pH 6.5) promotes dissociation of a significant portion of the organic acid to form H + and organic acid anions. H + is toxic to cells. By providing energy in the form of carbohydrates, constituents in the form of amino acids, appropriate temperatures and time for the cell to synthesize more proton pumps (right side of fig. 2), the cell can better adapt to low pH values and then better equip to cope with the excess protons by pumping them out.
In some embodiments, the food product comprises a fruit juice beverage, a carbonated beverage, a fermented beverage, fortified water, a fruit smoothie, a vegetable juice, a dairy smoothie, a dairy beverage, an energy beverage, a bagged beverage (sachet), a small serving beverage (shot), or a yogurt product. "servlet" refers to a liquid product formulation having a volume of about 4 ounces or less. In some embodiments, the food product is a "snack food," which refers to a ready-to-eat food product that does not require further cooking. In some embodiments, the snack produced requires refrigeration. It should be understood that food products according to the present disclosure may have any of a number of different specific formulations or components.
In some embodiments, the food product is a beverage. Beverages include, for example, juice beverages (e.g., beverages comprising one or more fruit juices and/or one or more vegetable juices), fountain beverages (e.g., fountain beverages with added electrolytes), frozen or chilled beverages, caffeine beverages, carbonated beverages, non-carbonated beverages, zero-to low-calorie beverages such as diet beverages or other reduced-calorie beverages (e.g., 0-150 kcal and up to 10 grams of sugar per 12 ounces (oz.)), and syrups or concentrates. Non-limiting examples of suitable dairy-containing beverages include milk (e.g., 2% milk) and other milk-containing beverages (e.g., milk-containing coffee drinks). Other suitable dairy-containing beverages include any beverage known in the art. In some embodiments, the food product is a beverage that may require refrigeration. In some embodiments, the food product is a beverage that is stable and safe to store under ambient conditions that do not require refrigeration. In some embodiments, the food product has a low pH of less than about 4.5. Suitable beverages may comprise sweeteners, water, dairy products, caffeine, carbonic acid, fruit juices, vegetable juices, food grade acids or mixtures thereof. Beverage products disclosed here include ready-to-drink liquid formulations (i.e., ready-to-drink beverages), beverage concentrates, beverage capsules (pods), and the like. The term "ready-to-drink" refers to a beverage that is formulated to be ingested as is. Thus, in some embodiments, the ready-to-drink beverage does not require dilution or addition prior to ingestion by the consumer. It should be understood that beverages and beverage concentrates according to the present disclosure may have any of a number of different specific formulations or compositions.
Examples
750g of the probiotic in bags were kept in a refrigerator at-80 ℃ until ready for use. The sample was then removed from the refrigerator and thawed by standing in a storage environment at 4 ℃ for 16 hours. The thawed probiotic was mixed with pasteurized single strength (single strength) apple juice cooled to 4 ℃ prior to mixing. The probiotic apple juice mixture had about 9.7 x 1010CFU/mL (colony forming units (CFU) per milliliter (mL)) of live probiotic cells (in a ratio of about 1:1 for the starting concentrated stock). After preconditioning the mixture at 4 ℃ for 2 hours in one embodiment, or 4 hours in another embodiment, the probiotic cells are seeded into a 32 ounce (946mL) strawberry banana juice base to reach about 108CFU/mL live probiotic cells. As a control, another sample of strawberry banana juice base was mixed with the same amount of non-preconditioned probiotic cells. For the control sample, an amount of non-preconditioned probiotic cells at the same level (e.g., about 108CFU/mL live probiotic cells) as the inoculated sample containing the preconditioned mixture was added to the strawberry banana juice base. After inoculation, the probiotic strawberry banana juice of both examples and one control sample was stored at 4 ℃. The amount of live probiotic cells in the inoculated juice was measured immediately after inoculation and then after storage at 4 ℃ for 2 days, 14 days, 30 days, 42 days, 65 days and 72 days.
FIG. 3 shows the average log CFU/mL measurements of live probiotics over each time interval. In all three samples, the rate of depletion of viable probiotic cells was gradually changed over a 65 day period. However, the rate of loss seen in the non-preconditioned control sample was greater than the preconditioned sample. For example, after 65 days, the percentage of colony forming units remaining in the initial CFU in the preconditioned sample was twice the percentage remaining in the non-preconditioned control sample. The results of preconditioning the samples indicate that the initial amount of probiotic cells introduced into the substrate can be reduced to achieve a product that meets the requirements of providing 10 billion CFU of probiotic cells in 8 ounces of product (236mL) prior to the shelf life date.
Other embodiments
The following illustrative embodiments are provided as further support for the disclosed invention:
in a first embodiment, the novel aspects described in the present disclosure relate to a method of forming stable probiotic cells, the method comprising: introducing the thawed probiotic cells into a preconditioned environment comprising sufficient nutrients to prepare the thawed probiotic cells for incorporation into a food product; and preconditioning the thawed probiotic cells in a preconditioning environment for at least about 2 hours, thereby producing preconditioned probiotic cells.
In another aspect of the first embodiment, a method of forming a stable probiotic cell comprises: introducing the thawed probiotic cells into a preconditioned environment comprising sufficient nutrients to prepare the thawed probiotic cells for incorporation into a food product; preconditioning the thawed probiotic cells in a preconditioning environment for at least about 2 hours, thereby producing preconditioned probiotic cells; and further comprising one or more of the following limitations selected from:
wherein the preconditioned environment comprises apple juice;
wherein the preconditioning environment comprises a fluid, and wherein the fluid and the probiotic cells are blended to achieve a predetermined probiotic concentration;
wherein the predetermined probiotic concentration is about 1.0 x 1010CFU/mL to 1.0 x 1011 CFU/mL;
wherein the preconditioning environment comprises a temperature of about 0 ℃ to about 39 ℃;
wherein the preconditioning environment comprises a temperature of about 0 ℃ to about 10 ℃;
wherein the incubation period is less than one complete cell division cycle;
wherein at least one complete cell division cycle is less than 24 hours;
thawing the frozen probiotic cells prior to the introducing step;
thawing the frozen probiotic cells at a temperature of about 0 ℃ to 39 ℃;
thawing the frozen probiotic cells for at least 8 hours;
after the providing step, adding the pre-treated probiotic cells to the pasteurized product matrix to produce a food product comprising stabilized probiotic cells;
wherein the nutrient comprises a carbohydrate;
wherein the nutrients comprise amino acids;
wherein the amino acid comprises homocysteine, cystathionine, cysteine or combinations thereof.
In a second embodiment, the novel aspect of the present disclosure relates to a preconditioned probiotic blend for incorporation into a pasteurized product matrix to provide a food product, the preconditioned probiotic blend comprising: a predetermined amount of thawed probiotic cells; a preconditioning fluid, wherein the preconditioning fluid comprises a nutrient; and a probiotic concentration of from about 1.0 x 1010CFU/mL to about 1.0 x 1011 CFU/mL.
In another aspect of the second embodiment, the novel aspects of the present disclosure relate to a preconditioned probiotic blend for incorporation into a pasteurized product matrix to provide a food product, the preconditioned probiotic blend comprising: a predetermined amount of thawed probiotic cells; a preconditioning fluid, wherein the preconditioning fluid comprises a nutrient; and a probiotic concentration of from about 1.0 x 1010CFU/mL to about 1.0 x 1011CFU/mL, the pre-conditioned probiotic blend for incorporation into a pasteurized product matrix to provide a food product further comprising one or more of the following limitations selected from:
wherein the pasteurized product base has a pH of less than about 7.0;
wherein the pasteurized product base has a pH of about 4.0 to about 5.0;
wherein the pasteurized product base has a first pH and the preconditioned probiotic blend has a second pH, wherein the first pH differs from the second pH by 1.0pH or less;
wherein the food is a snack food;
wherein the food product is a beverage;
wherein the beverage comprises a dairy product;
wherein the beverage comprises a fruit;
wherein the beverage comprises vegetables;
wherein the beverage comprises caffeine;
wherein the beverage comprises carbonation;
wherein at least 10 hundred million CFU's of probiotic cells are provided per 8 ounce (236mL) serving of the beverage;
wherein the beverage provides from about 1.6 x 108CFU/mL to about 2.6 x 108CFU/mL of viable probiotic;
wherein the nutrient comprises a carbohydrate;
wherein the nutrients comprise amino acids;
wherein the nutrients comprise organic acids;
wherein the preconditioning fluid is derived from a vegetable; and
wherein the pre-conditioning fluid is fruit-derived juice;
in another aspect of the invention, a food product (e.g., a beverage) comprises a preconditioned live probiotic blend comprising any combination or subcombination of the above-described features.
The words and phrases used herein should be understood and interpreted to have a meaning consistent with the understanding of those words and phrases by those skilled in the relevant art. No special definition of a term or phrase, i.e., a definition that is different from the ordinary and customary meaning as understood by those skilled in the art, is intended to be implied by consistent usage of the term or phrase herein. To the extent that a term or phrase is intended to have a special meaning, i.e., a meaning other than that understood by skilled artisans, such a special definition will be expressly set forth in the specification in a definitional manner that directly and unequivocally provides the special definition for the term or phrase.
The terms "comprising", "having" and variations thereof mean "including but not limited to", unless expressly specified otherwise. The term "comprising" when used in the appended claims, both original and modified, is intended to be inclusive or open-ended and does not exclude any additional unrecited elements, methods, steps or materials. The term "consisting of" excludes any element, step or material other than those specified in the claims. As used herein, "up to" includes zero, meaning that no amount (i.e., 0%) is added in some embodiments.
Unless specifically stated otherwise herein, the terms "a" and "an" and "the" are not limited to one such element, but rather mean "at least one. As used herein, the term "about" refers to the exact value indicated, as well as values within statistical variation or measurement error.
The methods disclosed herein may be suitably practiced in the absence of any element, limitation, or step not specifically disclosed herein. Similarly, particular product embodiments described herein may be obtained in the absence of any component not specifically described herein. Thus, the products described herein may comprise the listed components as those described above.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of 1 to 10 also incorporates a reference to all rational numbers within that range (i.e., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) as well as any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of all ranges explicitly disclosed herein are explicitly disclosed herein. These are only examples of what is specifically intended, and it is to be understood that all possible combinations of numerical values between the minimum and maximum values recited are explicitly stated in the application in a similar manner.
While the invention has been particularly shown and described with reference to several embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.