Cultured avocado cells and foods containing them

Abstract

This specification provides cultured avocado cells containing at least 15% by weight of protein, a method for producing the cultured avocado cells, and the use of the avocado cells in or as a food.

Description

Technical Field

[0001] The present disclosure generally relates to food. The present disclosure relates, without limitation, in particular to food-grade cultured avocado cells and foods containing the same.

Background Art

[0002] In this section, useful background information is illustrated without admitting that any of the techniques described herein represents the state of the art.

[0003] The cultivation of avocado fruits requires large amounts of resources such as water, the carbon footprint of avocado cultivation is considered high, and the conventional harvesting methods of avocado fruits may be seen as unsustainable. Additionally, the cultivation of avocado fruits is also associated with organized crime.

[0004] Therefore, from both environmental and human perspectives, there is a need to provide a more sustainable alternative to avocado fruits in food.

Summary of the Invention

[0005] The appended claims define the scope of protection. Examples and technical descriptions of devices, products and / or methods in the specification and / or drawings not included in the claims are presented as examples useful for understanding the invention, rather than as embodiments of the invention.

[0006] According to a first exemplary aspect, cultured avocado cells are provided, the cultured avocado cells containing at least 15% by weight of protein based on the total dry weight of the cultured avocado cells.

[0007] Preferably, the cultured avocado cells are food-grade avocado cells for human consumption.

[0008] Cultured avocado cells have not been proposed as a food or food ingredient. Surprisingly, cultured avocado cells can be used in or as a food and may have a beneficial macronutrient profile and / or a beneficial fatty acid profile. The cultured avocado cells of this invention have a significantly higher protein content compared to the mesocarp of a mature avocado fruit, based on their respective dry weight. This may result in health benefits, particularly compared to the mesocarp of a mature avocado fruit, and the energy content of cultured avocado cells may be reduced.

[0009] According to a second exemplary aspect of this specification, a method for producing cultured avocado cells is provided, the method is: The process of providing avocado cells, The process involves initiating cell suspension culture by seeding the provided avocado cells into a liquid medium, and culturing the avocado cells in cell suspension culture for a sufficient time and under conditions to result in a protein content of at least 15% by weight based on the total dry weight of the avocado cells. The process includes harvesting avocado cells from cell suspension culture to obtain cultured avocado cells.

[0010] Preferably, these cultured avocado cells are cultured avocado cells according to the first exemplary embodiment.

[0011] The method of the present invention makes it possible to produce avocado cells in a sustainable manner. Typically, the method is more sustainable and faster than cultivating avocado fruit, both from an environmental and human perspective. The method also requires fewer resources compared to avocado cultivation, potentially leading to cost savings, for example.

[0012] In a further exemplary embodiment, the Specified herein provides a food comprising cultured avocado cells according to the First Exemplary Embodiment, preferably comprising, based on the total weight of the food, 0.1 to 100% by weight, more preferably 0.5 to 90% by weight, more preferably 1.0 to 80% by weight, even more preferably 5 to 70% by weight, and most preferably 10 to 65% by weight of cultured avocado cells according to the First Exemplary Embodiment.

[0013] Although the cultured avocado cells of the present invention do not have the appearance and / or texture or structure of the mesocarp of an avocado fruit, they can be used as a substitute for or alternative to the mesocarp of an avocado fruit, particularly mashed avocado fruit, in foods such as guacamole and / or smoothies. The cultured avocado cells of the present invention may have a pleasant taste with the flavor characteristics of avocado.

[0014] Various non-binding exemplary embodiments and models have been described above. The embodiments described herein are used solely to illustrate selected embodiments or processes that may be used in various implementations. Some embodiments may be presented with reference only to specific exemplary models. It should be understood that corresponding embodiments may also apply to other exemplary models. [Brief explanation of the drawing]

[0015] Several exemplary embodiments are described with reference to the accompanying drawings.

[0016] [Figure 1] The content of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, and sterols in freeze-dried cultured avocado cells according to exemplary embodiments, depending on the culture time, is shown. [Figure 2] The analyzed fatty acid content of dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments is shown. [Figure 3]In addition to the analyzed sterol content of freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments, the analyzed tyramine, 2-pentadecylfuran, and 2-heptadecadienylfuran content is shown. [Figure 4] The analyzed content of benzaldehyde, trans-2-nonenal, and 1-octanol in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments is shown. [Figure 5] The analyzed content of 1-hexanol, 3-hexen-1-ol, tetradecane, and 3-octen-2-one in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments is shown. [Figure 6] The analyzed content of 2-octenal, 1-octen-3-ol, furfural, and copaene in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments is shown. [Figure 7] The analyzed content of hexanal, dodecane, 2-hexenal, 2-pentylfuran, 1-pentanol, and 2-heptanal in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments. [Figure 8] The analyzed contents of acetaldehyde, methanol, ethanol, and pentanal in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments are shown. [Figure 9] The analyzed content of isopropyl myristate, octanoic acid, and 2-methoxy-4-vinylphenol in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments. [Figure 10]The analyzed content of isopropyl palmitate, 2,4-di-tert-butylphenol, and diethyl phthalate in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments. [Figure 11] The analyzed content of 2-octenal-2-butyl, 2,4-decadienal, hexanoic acid, benzyl alcohol, and caryophyllene oxide in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments. [Figure 12] The analyzed content of trans-α-bergamotene and caryophyllene in freeze-dried cultured avocado cells and freeze-dried avocado fruit mesocarp according to exemplary embodiments is shown. [Figure 13] Qualitative catechin and procyanidin chromatograms of cultured avocado cells according to an exemplary embodiment are shown. [Figure 14] A photograph of a food product containing cultured avocado cells according to an exemplary embodiment is shown. [Figure 15] The analyzed individual amino acid content of cultured avocado cells according to an exemplary embodiment is shown. [Modes for carrying out the invention]

[0017] [Detailed explanation] Avocado fruit is typically defined as a berry rich in fatty acids, vitamins, minerals, dietary fiber, and antioxidants. As used herein, avocado or avocado fruit preferably refers to Persea americana unless otherwise specified, and avocado fruit mesocarp refers to the mesocarp of a mature avocado fruit. The development of an avocado fruit typically takes 6 to 12 months from flowering to maturity. The method described herein enables the production of cultured avocado cells much more rapidly, within a few days, a week, or two weeks. Furthermore, the method of the present invention may be considered more sustainable from both an environmental and human perspective compared to cultivating avocado fruit.

[0018] A mature avocado fruit typically contains 10–20% by weight of lipids, based on the total weight of the mesocarp. Based on the total dry weight of the mesocarp, the lipid content of a mature avocado fruit can be 60–70% by weight. The main lipids in a mature avocado fruit are triglycerides (TG), also known as triacylglycerols (TAG) or triacylglycerides. Typically, the lipids in a mature avocado fruit consist almost entirely of neutral lipids or TAGs (typically about 96% by weight of the total lipid weight), phospholipids, and glycolipids.

[0019] Compared to other fruits and / or berries, avocados have a high lipid content. The high lipid or fat content in the mesocarp of avocados contributes to the relatively high energy content of the mesocarp of a mature avocado, which is approximately 198 kcal / 100g or 829 kJ / 100g. As used herein, fat includes oil.

[0020] Typically, the mesocarp of a mature avocado fruit contains approximately 2% by weight of protein and less than 10% by weight of carbohydrates, and even less than 1% by weight of carbohydrates, based on the total weight of the mesocarp. Typically, the soluble protein content in the mesocarp increases during the maturation of the avocado fruit. Carbohydrate content is high during the early stages of fruit growth (sometimes exceeding 40% by weight of the total mesocarp weight), but decreases during maturation.

[0021] This specification provides cultured avocado cells, which contain at least 15% by weight of protein based on the total dry weight of the cultured avocado cells. Preferably, the cultured avocado cells contain 15-30% by weight, preferably 18-30% by weight, more preferably 20-30% by weight, and even more preferably 25-30% by weight of protein based on the total dry weight of the cultured avocado cells.

[0022] Several methods are known for evaluating protein content. For example, protein content may be determined by total amino acid analysis using ULC analysis of a hydrolyzed sample, as described in Example 5 of this disclosure, or by the Kjeldahl method according to NMKL 6:2003, as referred to in Example 3.1 of this disclosure. Preferably, in relation to this disclosure, protein content is determined by total amino acid analysis using ULC analysis of a hydrolyzed sample, more preferably as described in Example 5 of this disclosure. Although the Kjeldahl method is sometimes considered a standard method for evaluating protein content in the food industry, it may result in a higher ("overestimation") of protein content compared to so-called direct amino acid analysis methods, such as total amino acid analysis by ULC analysis of a hydrolyzed sample.

[0023] Preferably, when the protein content is determined by total amino acid analysis using ULC analysis of a hydrolyzed sample, as particularly defined in Example 5, the cultured avocado cells contain at least 15% by weight, preferably 15-30% by weight, more preferably 15-25% by weight, more preferably 18.0-25.0% by weight, and even more preferably 18.0-22.0% by weight of protein, based on the total dry weight of the cultured avocado cells. Preferably, when the protein content is determined by the Kjeldahl method, in accordance with NMKL 6:2003, as referred to, for example in Example 3.1, the cultured avocado cells contain at least 15% by weight, preferably 15-30% by weight, more preferably 20-30% by weight, and even more preferably 25-30% by weight of protein, based on the total dry weight of the cultured avocado cells. In this regard, the concept of containing a certain amount of protein refers to the reported protein content as determined by the respective analytical method.

[0024] Surprisingly, the cultured avocado cells of the present invention contain more protein than the mesocarp of an avocado fruit, based on their respective dry weights. The protein content of the cultured avocado cells of the present invention can be more than three times that of the mesocarp of an avocado fruit, based on their respective dry weights and as shown in the experimental examples of this disclosure. "Based on their respective dry weights" means, in relation to this disclosure, a comparison of values ​​calculated based on the total dry weight of each sample in question (cultured avocado cells, mesocarp of an avocado fruit). The increased protein content may result in a beneficial nutritional or macronutrient profile, enabling its use as a plant-based or plant-derived protein source, and / or resulting in an increase in protein content in food, for example. Preferably, the cultured avocado cells of the present invention are food-grade avocado cells for human consumption.

[0025] Surprisingly, it was found that the cultured avocado cells of the present invention may contain one or more major nutrients (fat or lipids, proteins, and / or carbohydrates) in different proportions than the mesocarp of a mature avocado fruit.

[0026] Preferably, the cultured avocado cells of the present invention contain more protein and less lipid than the mesocarp of a mature avocado fruit. Therefore, in certain preferred embodiments, the cultured avocado cells contain 0.5 to 10% by weight, preferably 1 to 7% by weight, more preferably 1.5 to 6% by weight, and even more preferably 2 to 5% by weight of lipids, based on the total dry weight of the cultured avocado cells. In these embodiments, the protein content of the cultured avocado cells is preferably as defined above. These lipid contents are much lower than the typical lipid content of a mature avocado fruit. In these embodiments, the energy content of the cultured avocado cells is much lower compared to the mesocarp of a mature avocado fruit. Therefore, the cultured avocado cells of the present invention can provide a high-protein, low-fat, and low-energy alternative to avocado fruit.

[0027] The cultured avocado cells of the present invention may have a higher carbohydrate content compared to the mesocarp of a mature avocado fruit. In certain embodiments, the cultured avocado cells contain 10-65% by weight, preferably 14-65% by weight, more preferably 25-64% by weight, and even more preferably 55-64% by weight of carbohydrates, based on the total dry weight of the cultured avocado cells. In these embodiments, the protein content, lipid content, or both of the cultured avocado cells are preferably as defined above. Typically, a higher carbohydrate content contributes to a lower total energy content of the cultured avocado cells compared to the mesocarp of a mature avocado fruit.

[0028] The lipid content and / or carbohydrate content are preferably analyzed taking into account the moisture content, as described in Example 3.1 of this disclosure.

[0029] The lipid composition of the cultured avocado cells of the present invention may be considered beneficial from a nutritional and / or health standpoint. The proportion of polyunsaturated fatty acids (PUFAs), when standardized by total lipid content, is surprisingly much higher, up to four times higher, compared to the proportion of PUFAs in the mesocarp of a mature avocado fruit. PUFAs are generally considered particularly desirable in food, for example, due to their associated health benefits. When standardized by their respective total lipid content, the cultured avocado cells of the present invention may have a lower proportion of monounsaturated fatty acids (MUFAs) and / or a higher content of saturated fatty acids (SAFAs) compared to the mesocarp of a mature avocado fruit.

[0030] In certain preferred embodiments, the cultured avocado cells contain 30-70% by weight, preferably 40-65% by weight, and more preferably 50-60% by weight of polyunsaturated fatty acids, based on the total weight of lipids in the cultured avocado cells. Optionally, the cultured avocado cells contain 5-15% by weight, preferably 8-12% by weight, of monounsaturated fatty acids, and / or 20-40% by weight, preferably 25-40% by weight, and more preferably 30-35% by weight, of saturated fatty acids, based on the total weight of lipids in the cultured avocado cells.

[0031] In the cultured avocado cells of the present invention, the monounsaturated oleic acid (C18:1) content may be lower compared to the mesocarp of an avocado fruit. The main fatty acids in the cultured avocado cells of the present invention are typically polyunsaturated linoleic acid (C18:2n-6) and α-linoleic acid (C18:3n-3), and the linoleic acid (C18:2n-6) content is typically higher than the α-linoleic acid (C18:3n-3) content. Therefore, in cultured avocado cells, the linoleic acid content may be higher compared to the mesocarp of an avocado fruit. In certain embodiments, cultured avocado cells contain 30–55% by weight, preferably 35–50% by weight, more preferably 40–50% by weight of linoleic acid (C18:2n-6) and / or 5–15% by weight or 6–11% by weight of α-linoleic acid (C18:3n-3), and optionally 5–10% by weight of oleic acid (C18:1), and optionally 20–30% by weight or 20–25% by weight of palmitic acid (C16:0), based on the total weight of lipids in the cultured avocado cells. These values ​​differ from those typical of the mesocarp of a mature avocado fruit. Preferably, cultured avocado cells may contain substantially no palmitoleic acid (C16:19) or have a palmitoleic acid (C16:19) content below the detection limit, as determined by direct in situ transesterification and gas chromatography, for example, as described in Glaser et al. 2010 High-Throughput Analysis of Total Plasma Fatty Acid Composition with Direct In Situ Transesterification, PLOS ONE (https: / / doi.org / 10.1371 / journal.pone.0012045).

[0032] The total lipid content of the cultured avocado cells of the present invention is typically significantly lower than that of the mesocarp of a mature avocado fruit, thus providing certain benefits. For example, a higher amount of PUFAs can result in a lower total lipid or fat content, and consequently a lower energy content. Preferably, the cultured avocado cells contain at least 5 mg / g, preferably 5-25 mg / g, more preferably 7-20 mg / g, more preferably 10-18 mg / g, and even more preferably 10-15 mg / g of polyunsaturated fatty acids, based on the total dry weight of the cultured avocado cells. Preferably, the cultured avocado cells contain at least 5 mg / g, for example 5-20 mg / g, preferably at least 8 mg / g, more preferably 8-15 mg / g, and even more preferably 8-10 mg / g of linoleic acid (C18:2n6), and / or 1 mg / g or more, preferably 1.5 mg / g or more, more preferably 1-3 mg / g, and even more preferably 1.5-2 mg / g of alpha-linoleic acid (C18:3n3), based on the total dry weight of the cultured avocado cells. These amounts are preferably surprisingly high considering the typical total lipid content of the cultured avocado cells of the present invention, as defined above. The amount of saturated fatty acids, which are generally considered less healthy than unsaturated fatty acids, can also be kept at a low level when the total lipid or fat content is low. In certain embodiments, the cultured avocado cells contain 3-15 mg / g, preferably 5-10 mg / g of saturated fatty acids, based on the total dry weight of the cultured avocado cells. The amount of such saturated fatty acids is, in fact, much less than the SAFA content in the mesocarp of a mature avocado fruit, based on their respective dry weights. However, when the proportion of SAFA is standardized against the total lipid content, it can be higher in the cultured avocado cells of the present invention. In certain embodiments, the cultured avocado cells contain less than 10 mg / g or less than 5 mg / g, preferably 1 to 3 mg / g, of monounsaturated fatty acids, based on the total dry weight of the cultured avocado cells, and optionally less than 5 mg / g, preferably up to 2 mg / g, of oleic acid (C18:1). Such amounts are much less than the amounts contained in the mesocarp of a mature avocado fruit, based on their respective dry weights.

[0033] The free fatty acid (FFA) content may be similar between cultured avocado cells and the mesocarp of avocado fruit, based on their respective dry weights. The cultured avocado cells of the present invention may contain 0.1 to 1.0 mg / g, preferably 0.2 to 0.5 mg / g of FFA, based on the total dry weight of the cultured avocado cells.

[0034] The sterol content in cultured avocado cells of the present invention may be higher than that in the mesocarp of a mature avocado fruit. In certain preferred embodiments, cultured avocado cells contain 0.5 to 2.0 mg / g, preferably 0.5 to 1.5 mg / g, and more preferably 0.75 to 1.25 mg / g of sterols, based on the total dry weight of the cultured avocado cells. In particular, the β-sitosterol content in cultured avocado cells may be higher than that in the mesocarp of an avocado fruit. For example, cultured avocado cells may contain 0.5 to 1.0 mg / g of β-sitosterol, based on the total dry weight of the cultured avocado cells.

[0035] In connection with this disclosure, the fatty acid content is preferably determined according to the method reported by Glaser et al. 2010 High-Throughput Analysis of Total Plasma Fatty Acid Composition with Direct In Situ Transesterification, PLOS ONE (https: / / doi.org / 10.1371 / journal.pone.0012045), unless otherwise specified. The FAME, FFA, and sterol content is preferably determined, in connection with this disclosure, according to the method described by Seppanen-Laakso et al. 2017 UPLC-ELSD Analysis of Algal Lipid Classes and Derivatization of Bound and Free Fatty Acids and Sterols for GC-MS Methods, Biofuels from Algae, vol. 1980, pp. 223-232 (DOI: 10.1007 / 7651_2017_109), respectively.

[0036] Compounds in avocado fruit that contribute to the typical flavor of the mesocarp include pentanal, (E)-2-pentenal, ethyl acetate, benzaldehyde, hexanal, and (E)-2-hexenal. Of these, pentanal, (E)-2-pentenal, ethyl acetate, and benzaldehyde are thought to contribute to, or give rise to, the typically pleasant nutty, fruity flavor. Hexanal and (E)-2-hexenal are thought to contribute to, or provide to, oily and grassy aromas, which, while typical of the avocado mesocarp, can be considered less appealing and even unpleasant.

[0037] The cultured avocado cells of the present invention may contain benzaldehyde and / or pentanal, preferably both, which, based on their respective dry weights, are present in amounts clearly greater than those in mature avocado mesocarp and provide or contribute to a pleasant fruity and nutty flavor. This can result in a stronger and / or more pleasant taste compared to the mesocarp of mature avocado fruits. Thus, in certain preferred embodiments, the cultured avocado cells preferably contain, as determined by gas chromatography-mass spectrometry (GC-MS) as described in Example 3.0, at least 1.5·10 8 area / g, preferably 1.5·10 8 ~3.5·10 8 area / g, more preferably 2.0·10 8 ~3.0·10 8 area / g, even more preferably 2.2·10 8 ~3.0·10 8 area / g or 2.5·10 8 ~3.0·10 8 area / g of pentanal, and / or more than 6.0·10 8 area / g, preferably more than 6.5·10 8 ~1.5~10 9 area / g, more preferably 7.0·10 8 ~1.4·10 9 area / g, even more preferably 8.0·10 8 ~1.3·10 9 area / g, and even more preferably 1.0·10 9 ~1.3·10 9 area / g of benzaldehyde. The content of pentanal and / or benzaldehyde can be 1.5 times or more, and even 2 times or more, respectively, compared to their respective contents in the mesocarp of avocado fruits, based on their respective dry weights.

[0038] Hexanal and (E)-2-hexenal may be thought to contribute to or impart an unattractive flavor, but are thought to contribute to a flavor similar to that of the mesocarp of an avocado fruit. The cultured avocado cells of the present invention may contain more hexanal than the mesocarp of an avocado fruit, based on their respective dry weights. In certain embodiments, the cultured avocado cells may contain 3.10 as determined by GC-MS, preferably as described in Example 3.0, based on the total dry weight of the cultured avocado cells. 9 ~6.10 9 area / g or 4·10 9 ~5.5·10 9 The cultured avocado cells contain area / g of hexanal. In certain embodiments, it may be more important that the cultured avocado cells contain a higher amount of benzaldehyde and / or pentanal, preferably both, which are present within the range defined above, rather than a higher amount of hexanal, as this is thought to impart or contribute to a pleasant fruity and nutty flavor. Typically, the amount of 2-hexenal in the cultured avocado cells of the present invention is about the same as, or slightly more than, the amount in the mesocarp of an avocado fruit. The cultured avocado cells of the present invention contain about 0.5-10⁶ hexenal based on the total dry weight of the cultured avocado cells, as determined by GC-MS, preferably as described in Example 3.0. 9 It may contain 2-hexenal at a concentration of area / g or less. The stronger flavor of the cultured avocado cells of the present invention compared to the mesocarp of a mature avocado fruit may benefit from requiring less cultured avocado cells to produce the desired avocado-like flavor. This may (further) reduce resource consumption, food energy content, and costs. Surprisingly, fresh cultured avocado cells as described herein were found to have a pleasant flavor and aroma, and avocado flavor characteristics, as reported in Example 6 of this disclosure.

[0039] The mesocarp of avocado fruit contains various organic compounds, referred to herein as volatile substances. Many of the volatile substances found in the mesocarp of mature avocado fruit are also present in the cultured avocado cells described herein. However, the volatile substances and their content are typically not identical between the mesocarp of avocado fruit and the cultured avocado cells of the present invention.

[0040] In certain embodiments, the cultured avocado cells of the present invention may be substantially free of caryophyllene, or their caryophyllene content may be below the detection limit, as determined by GC-MS, preferably as described in Example 3.0. Since caryophyllene is considered a typical volatile substance in the mesocarp of avocado fruit, it is surprising that caryophyllene may be absent or present in very small amounts in cultured avocado cells. Similarly, the tocopherol or vitamin E content in cultured avocado cells may be below the detection limit, as determined by GC-MS, preferably as described in Example 3.0, or the cultured avocado cells may be substantially free of tocopherol. Since tocopherol is typically present in significant amounts in the mesocarp of avocado, it is surprising that it may be present in very small amounts or not at all in the cultured avocado cells of the present invention. Therefore, in certain embodiments, the cultured avocado cells are substantially free of caryophyllene and / or tocopherol, preferably substantially free of both.

[0041] The cultured avocado cells of the present invention may substantially contain one or more, or all, of 2-pentadecylfuran, 2-heptadecadienylfuran, 3-hexen-1-ol, caryophyllene oxide, and / or trans-α-bergamotene. These are typically found in the mesocarp of mature avocado fruit.

[0042] The content of one or more of dodecane, 2-heptenal, acetaldehyde, 1-hexanol, tetradecane, furfural, copaene, 1-octanol, and / or 2-heptadecadienylfuran, or all of these, may be lower in cultured avocado cells compared to the mesocarp of a mature avocado fruit, based on their respective dry weights. For example, 1-octanol may be preferable to reduce because it can cause or contribute to an unpleasant flavor.

[0043] In certain embodiments, cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 2.10. 8 area / g less than, preferably 0.1-10 8 ~1.5·10 8 area / g, comfort level 0.5·10 8 ~1.0·10 8 Contains area / g of acetaldehyde. The acetaldehyde content of cultured avocado cells may be less than half or two-fifths of that of the mesocarp of an avocado fruit, based on their respective dry weights.

[0044] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be between 1.0 and 3.5.10. 8 area / g less than, preferably 1.5-10 8 ~3.0·10 8 It contains area / g of 1-octanol. The 1-octanol content of cultured avocado cells may be less than 2 / 3 of that of the mesocarp of an avocado fruit, based on their respective dry weights.

[0045] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 9 area / g less than, preferably 0.1-10 9 ~1.0·10 9Comfort 0.1·10 9 ~0.5·10 9 Contains area / g of dodecane. Cultured avocado cells may contain less than one-fifth, or even less than one-tenth, of the dodecane compared to the mesocarp of an avocado fruit, based on their respective dry weights.

[0046] In certain embodiments, cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 1.10. 8 ~3.10 8 area / g, preferably 1.5-10 8 ~2.5·10 8 Contains area / g of 1-hexanol. The cultured avocado cells of the present invention may contain 1-hexanol in amounts less than half, or even less than one-third, of the amount of 1-hexanol found in the mesocarp of a mature avocado fruit, based on their respective dry weights.

[0047] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 9 ~1.3·10 9 Contains area / g of 2-heptenal. The cultured avocado cells of the present invention may contain 2-heptenal in amounts less than half, or even less than one-third, of the amount of 2-heptenal found in the mesocarp of a mature avocado fruit, based on their respective dry weights.

[0048] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 8 ~1.5·10 8 area / g, preferably 0.7-10 8 ~1.5·10 8 Tetradecane area / g, and / or 1.5·10 8 ~2.5·10 8 area / g contains 3-octen-2-one

[0049] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 8 ~2.10 8 area / g, preferably 0.7-10 8 ~1.5·10 8 area / g, comfortable 0.7·10 8 ~1.2.10 8 It contains furfural at area / g. The furfural content of cultured avocado cells may be less than half, or even about one-fifth or less, of the furfural content of the mesocarp of avocado fruit, based on their respective dry weights. The copaene content of cultured avocado cells is determined by GC-MS, preferably as described in Example 3.0, based on the total dry weight of cultured avocado cells, and is less than one-fifth, for example, 5-10, compared to cultured avocado cells, based on their respective dry weights. 8 area / g may be less than or equal to the specified area / g.

[0050] The content of one or more of the following, or all of them, may be higher in cultured avocado cells compared to the mesocarp of a mature avocado fruit, based on their respective dry weights: tyramine, ethanol, 2-pentylfuran, 2-octenal, 1-octen-3-ol, trans-2-nonenal, isopropyl myristate, octanoic acid, isopropyl palmitate, 2,4-di-tert-butylphenol, hexanoic acid, 2-octanal-2-butyl, and / or benzyl alcohol. Trans-2-nonenal may contribute to a slightly oily, fried taste with a nutty flavor background, and its content may be slightly higher in cultured cells compared to the mesocarp of avocado fruit, based on their respective dry weights.

[0051] In certain embodiments, cultured avocado cells are determined by GC-MS based on the total dry weight of cultured avocado cells, preferably as described in Example 3.0, to be 0.8-10. 9 area / g or more, preferably 0.9-109 ~2.10 9 area / g, comfort level 1.0·10 9 ~1.5·10 9 It contains area / g of 2-octenal. Cultured avocado cells may contain at least 1.5 times, and even more than 2 times, the amount of 2-octenal compared to the mesocarp of cultured avocado cells, based on their respective dry weight. 2-octenal can impart a sweet, oily, citrus peel-like flavor, and a higher 2-octenal content can result in a stronger such flavor.

[0052] Preferably, cultured avocado cells contain (one or more) catechins and / or (one or more) procyanidins, and more preferably (one or more) catechins and / or (one or more) oligomeric procyanidins. (One or more) catechins and (one or more) procyanidins, in particular (one or more) oligomeric procyanidins, are typically considered to be beneficial to health or to provide health benefits.

[0053] In certain embodiments, cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 3.5-10. 8 ~10·10 8 area / g, preferably 5.0·10 9 ~9.0·10 9 area / g of octanoic acid, and / or 0.5·10 8 ~3.0·10 8 area / g, preferably 1.0·10 8 ~2.0·10 8 Contains area / g of isopropyl myristate. The octanoic acid content and / or isopropyl myristate content may be 15 times, or even 20 times, more than, the mesocarp of an avocado fruit, based on their respective dry weights.

[0054] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 9 ~2.10 9 area / g, preferably 0.8-10 9 ~1.7·10 9 area / g, comfort level 1.0·10 9 ~1.5·10 9 It contains isopropyl palmitate. Cultured avocado cells may contain at least 5 times, and even more than 7 times, isopropyl palmitate compared to the mesocarp of cultured avocado cells, based on their respective dry weight.

[0055] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 9 ~2.10 9 area / g, preferably 0.7-10 9 ~1.7·10 9 area / g, more comfortably 0.8·10 9 ~1.5·10 9 It contains 2,4-di-tert-butylphenol. Cultured avocado cells may contain at least 8 times, and even more than 9 times, 2,4-di-tert-butylphenol compared to the mesocarp of cultured avocado cells, based on their respective dry weight.

[0056] In certain embodiments, the cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.5-10. 9 ~2.5·10 9 area / g, preferably 1.0·10 9 ~2.0·10 9 Contains area / g of benzyl alcohol. Cultured avocado cells may contain at least 50 times, and even 55 times or more, the amount of benzyl alcohol compared to the mesocarp of cultured avocado cells, based on their respective dry weight.

[0057] The cultured avocado cells were determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, to be 0.3-10. 8 area / g or greater, for example, 0.5·10 8 ~1.5·10 8 It may contain 2-octenal-2-butyl, such as area / g. The mesocarp of avocado fruit typically does not contain 2-octenal-2-butyl, or at least does not contain it in large quantities.

[0058] Cultured avocado cells are determined by GC-MS based on the total dry weight of the cultured avocado cells, preferably as described in Example 3.0, and are 5-10. 8 ~10·10 8 It may contain area / g of hexanoic acid. This is higher than the typical amount in the mesocarp of an avocado fruit, based on their respective dry weights. Hexanoic acid may contribute to the desired flavor of cultured avocado cells.

[0059] As illustrated above, the content of (one or more) specific volatile substances can vary significantly in cultured avocado cells compared to the mesocarp of an avocado fruit, based on their respective dry weights. The cultured avocado cells of the present invention may provide (one or more) specific compounds in (one or more) desired amounts and / or amounts not obtainable in the mesocarp of an avocado fruit.

[0060] This specification provides a method for producing cultured avocado cells, the method being described below. The process of providing avocado cells, The process involves initiating cell suspension culture by seeding the provided avocado cells into a liquid medium, and culturing the avocado cells in cell suspension culture for a sufficient time and under conditions to result in a protein content of at least 15% by weight based on the total dry weight of the avocado cells. The process includes harvesting avocado cells from cell suspension culture to obtain cultured avocado cells.

[0061] Preferably, the method is for producing cultured avocado cells as described herein, and preferably as described in any one of the embodiments described above. The culture of avocado cells may be carried out in one or more bioreactors.

[0062] Preferably, the step of providing avocado cells includes growing callus from avocado on a solid growth medium and providing avocado cells from the callus (and cell suspension culture is then initiated from these provided cells). The provided cells may be provided fresh from the callus or may be stored in liquid nitrogen at a temperature below -190°C, for example, before providing them to initiate cell suspension culture. Typically, frozen cells are thawed before initiating suspension culture.

[0063] Callus can be grown from avocado fruit material or from sections of avocado plants, such as (one or more) in vitro grown avocado plants. In certain embodiments, callus is grown from avocado seeds or (one or more) seed sections, preferably from mature avocado fruit (e.g., 6 to 12 months after flowering). Preferably, seed material may be an embryo or (one or more) parts thereof, and / or seeds may be germinated typically in water before callus is grown from them. Similarly, (one or more) seed sections may be provided, if desired, by cutting germinated seeds. Germination time may be, for example, 1 to 3 months, preferably about 2 months. In certain embodiments, callus is grown from sections of avocado plants, such as sterile stem sections, including small plants. In certain embodiments, callus can be grown from material from the inner layer between the pericarp and mesocarp of an avocado fruit.

[0064] In certain preferred embodiments, the method of the present invention includes a step of sterilizing, preferably surface sterilizing, one or more parts of the avocado on which the callus is to be grown, before the callus is to grow. Suitable sterilization methods include, for example, surface sterilization with 70% ethanol, a surfactant (e.g., Tween 80), and / or sodium hypochlorite (1-10% v / v), followed by washing with sterile water. However, any suitable sterilization or surface sterilization method may be used.

[0065] Typically, callus is grown on a solid growth medium from avocado, preferably avocado fruit material, (one or more) plant sections, and / or (one or more) sections from (one or more) seeds as defined above. The solid growth medium may contain plant growth regulators, preferably at least one cytokinin or cytokinin-like plant growth regulator (CN) and at least one auxin (AUX). Examples of suitable CNs and suitable AUXs are mentioned below.

[0066] The process of establishing or growing callus from avocado typically involves incubating a portion of avocado, preferably a seed or (one or more) seed sections, on a solid medium containing nutrients and plant growth regulators. For example, the solid medium may be Murashige and Skoog medium (MS medium as described in Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15, 473-497) solidified with a suitable coagulant such as agar or Gelrite®. For example, the growth medium may be solidified with 6.5 g / l agar or 3 g / l Gelrite®, but any suitable coagulant in an appropriate amount may be used. Typically, (one or more) sufficiently pure coagulants are used because impurities can hinder growth.

[0067] The solid medium may contain, for example, 2 - 3% w / v, preferably about 3% w / v sucrose. The plant growth regulator may include at least one cytokinin or cytokinin - like plant growth regulator (CN) and at least one auxin (AUX). The wt:wt ratio of CN to AUX may be in the range of 1:100 to 1:2. The CN concentration may be 0.1 - 5.0 ppm and the AUX concentration may be 1.0 - 10 ppm. Examples of suitable CNs include kinetin, 6-(γ,γ - dimethylallylamino) purine, zeatin, or any combination thereof, and examples of suitable AUXs include α - naphthaleneacetic acid, indoleacetic acid, picloram, or any combination thereof, preferably α - naphthaleneacetic acid, indoleacetic acid, or any combination thereof. More preferably, CN is kinetin in an amount preferably of 0.1 - 5.0 ppm, more preferably about 0.1 ppm, and AUX is α - naphthaleneacetic acid (NAA) in an amount preferably of 1.0 - 10 ppm, more preferably about 1.0 ppm.

[0068] The incubation temperature for establishing callus can be within the range of 17 - 28°C, for example 22 ± 2°C. The incubation can be carried out in the dark and / or in the light. Typically, the incubation includes incubation in both the dark and the light, such as alternating light and dark periods according to a predetermined regime. Preferably, preferably using normal light with an irradiation of 30 - 60 μmol / m 2 s for the daytime and / or light period, a 16 - hour light period: 8 - hour dark period day: night photoperiod is applied.

[0069] The resulting soft callus can be transferred to a fresh medium. Preferably, the fresh medium is a solid medium containing the plant growth regulators or nutrients described above. The resulting callus can be maintained and cultivated using the plant growth regulators or nutrients described above and a solid medium containing the plant growth regulators, preferably using a lighting regime described above. If desired, the callus can be maintained in light (no dark period) or in darkness (no light period). The callus can be maintained at a temperature in the range of 17-28°C, such as 24°C. The temperature at which the callus is maintained may be approximately the same as or different from the temperature at which it is established.

[0070] Preferably, the callus culture is subcultured every 3 to 5 weeks. The subculture conditions may be the same as those described above for establishing or growing the callus.

[0071] The step of providing avocado cells may preferably include the step of selecting avocado cells from callus, which is preferably obtained as described above. By appropriately selecting the cells in which cell suspension culture is initiated, and / or the conditions and / or duration of cell suspension culture, a desired content of the desired compound in the cultured cells can be obtained, while the presence of certain compounds in the cultured cells can be limited or avoided as desired. As those skilled in the art will understand, cultured cells typically differ in composition and / or properties from, for example, naturally occurring fruit or berry cells.

[0072] Typically, avocado cells, particularly preferably from callus obtained as described above, produce major nutrients (proteins, lipids or fats, and carbohydrates) in cell suspension culture. However, if required or desired, the method of the present invention may include a selection step of selecting avocado cells that produce proteins, lipids, and / or carbohydrates, preferably at least proteins, more preferably proteins, lipids, and carbohydrates, in cell suspension culture, wherein the avocado cells are preferably selected from callus obtained as described above.

[0073] In certain preferred embodiments, the step of providing avocado cells is: The process involves selecting avocado cells that produce (one or more) catechins and / or (one or more) procyanidins, preferably (one or more) catechins and / or (one or more) oligomeric procyanidins, more preferably both, in cell suspension culture. The process includes the steps of providing selected avocado cells (and initiating a cell suspension culture from these cells). (One or more) catechins and / or (one or more) procyanidins, in particular (one or more) oligomeric procyanidins, may provide health benefits.

[0074] In certain preferred embodiments, the step of providing avocado cells is: The process involves selecting avocado cells that produce benzaldehyde and / or pentanal, preferably both, and optionally hexanal and / or (E)-2-hexenal, in a cell suspension culture. The process includes providing selected avocado cells (and initiating cell suspension culture from these cells). This may result in the flavor benefits described above.

[0075] In certain preferred embodiments, the step of providing avocado cells is: A step of selecting avocado cells in a cell suspension culture that are essentially free from one or more or all of 2-pentadecylfuran, 2-heptadecadienylfuran, 3-hexen-1-ol, caryophyllene oxide, trans-α-bergamotene, caryophyllene, and / or tocopherol, preferably free from at least caryophyllene and / or tocopherol, more preferably free from at least both, wherein the avocado cells are preferably selected from callus obtained as described above. The process includes the steps of providing selected avocado cells (and initiating cell suspension culture from these cells).

[0076] In a particular preferred embodiment, the step of providing avocado cells is a step of selecting avocado cells in a cell suspension culture that produce polyunsaturated fatty acids, preferably at least linoleic acid (C18:2n-6) and / or α-linoleic acid (C18:3n-3), preferably both, without substantially producing palmitoleic acid (C16:19) as desired, wherein the avocado cells are preferably selected from the callus obtained as described above. The process includes the steps of providing selected avocado cells (and initiating cell suspension culture from these cells).

[0077] In a particular preferred embodiment, the step of providing avocado cells is a step of selecting avocado cells that produce sterols, preferably at least β-sitosterol, in a cell suspension culture, wherein the avocado cells are preferably selected from the callus obtained as described above. The process includes the steps of providing selected avocado cells (and initiating cell suspension culture from these cells).

[0078] By selecting cells that produce (one or more) specific compounds in cell suspension culture, it becomes possible to obtain cultured cells containing these compounds in amounts potentially greater than those found in the mesocarp of an avocado fruit. By selecting cells that substantially do not produce (one or more) specific compounds or that substantially do not contain (one or more) specific compounds in cell suspension culture, it becomes possible to obtain cultured cells that are substantially free of (one or more) such compounds naturally, or that contain (one or more) such compounds, for example, in amounts below the detection limit. "Naturally free" means, as used herein, that it is not necessary to remove (one or more) of the above compounds in order to obtain cultured cells that are substantially free of these compounds.

[0079] Depending on the cells selected as described above, cells with a composition significantly different from that of the mesocarp of an avocado fruit may be produced in cell suspension culture.

[0080] It is particularly preferable that the provided avocado cells, from which cell suspension culture is initiated, are selected so as not to produce, and not essentially contain, the compounds defined in the combination of embodiments described above.

[0081] Preferably, the cultured avocado cells of the present disclosure can be obtained by the methods of the present disclosure or in accordance with any one of the embodiments of the present disclosure.

[0082] In the method of the present invention, a cell suspension culture is initiated by seeding provided avocado cells, preferably callus, preferably cells from callus obtained as described above, and optionally appropriately selected cells as described above, into a liquid medium, and cultured avocado cells are obtained by harvesting the avocado cells from the cell suspension culture. In the method of the present invention, the avocado cells are cultured in the cell suspension culture for a sufficient time and under conditions to result in a protein content of at least 15% by weight, preferably 15-30% by weight, more preferably 18-30% by weight, more preferably 20-30% by weight, and even more preferably 25-30% by weight, based on the total dry weight of the avocado cells.

[0083] Surprisingly, the protein content of cultured avocado cells is obtained by adding glycine (Gly) and glutamic acid (Glu) to the liquid medium, preferably at 0.6-5.0 mM (millimoles / dm³). 3 It has been found that the amount can be further increased by including a total amount of preferably 0.8 to 3.0 mM, more preferably 1.0 to 2.0 mM. Preferably, there is more Glu than Gly in the liquid medium. In certain preferred embodiments, the liquid medium contains 0.01 to 0.05 mM Gly and 0.5 to 1.5 mM Glu.

[0084] As discussed above, preferably, when the protein content is determined by total amino acid analysis using ULC analysis of hydrolysis samples, as defined in Example 5, the cultured avocado cells contain at least 15% by weight, preferably 15-30% by weight, more preferably 15-25% by weight, more preferably 18.0-25.0% by weight, and even more preferably 18.0-22.0% by weight of protein, based on the total dry weight of the cultured avocado cells. Preferably, when the protein content is determined by the Kjeldahl method according to NMKL 6:2003, as mentioned in Example 3.1, for example, the cultured avocado cells contain at least 15-30% by weight, preferably 20-30% by weight, and even more preferably 25-30% by weight of protein, based on the total dry weight of the cultured avocado cells.

[0085] Conditions of cell suspension culture, such as temperature, agitation, culture medium, nutrients and their amounts and regimes, inducers and their amounts and regimes (including the absence of one or more inducers), and light and / or dark periods, typically affect the production of compounds in cultured cells, as understood by those skilled in the art, and can decrease or increase the production of (one or more) specific compounds. The duration of culture can affect the concentration of at least specific compounds in cultured cells, as understood by those skilled in the art. The relationship between culture time and the concentration of a specific compound is not necessarily linear, and the concentration of a specific compound may peak within a particular culture time range, after which its concentration may begin to decrease or remain somewhat constant. Those skilled in the art will understand that the conditions and / or duration of cell suspension culture can be appropriately selected to result in cultured avocado cells of the present disclosure and / or any embodiment thereof, especially if the provided cells are from callus obtained as described above and / or appropriately selected callus as described above.

[0086] Preferably, avocado cells are cultured in cell suspension culture for a sufficient time and under conditions to result in a lipid content of 0.5–10% by weight, preferably 1–7% by weight, more preferably 1.5–6% by weight, and even more preferably 2–5% by weight, based on the total dry weight of the cultured avocado cells, and / or a carbohydrate content of 10–65% by weight, preferably 14–65% by weight, more preferably 25–64% by weight, and even more preferably 55–64% by weight.

[0087] In certain preferred embodiments, avocado cells are cultured in cell suspension culture for a sufficient time and under conditions to result in a polyunsaturated fatty acid content of at least 5 mg / g, preferably 5-25 mg / g, more preferably 7-20 mg / g, more preferably 10-18 mg / g, and even more preferably 10-15 mg / g, based on the total dry weight of cultured avocado cells, and a linoleic acid (C18:2n6) content of at least 5 mg / g, for example 5-20 mg / g, preferably at least 8 mg / g, more preferably 8-15 mg / g, and even more preferably 8-10 mg / g, and / or an α-linoleic acid (C18:3n3) content of 1 mg / g or more, preferably 1.50 mg / g or more, more preferably 1-3 mg / g, and even more preferably 1.5-2 mg / g. In certain embodiments, avocado cells are cultured in cell suspension culture for a time and under conditions that result in a saturated fatty acid content of 3–15 mg / g, preferably 5–10 mg / g, and / or a monounsaturated fatty acid content of less than 10 mg / g or less than 5 mg / g, preferably 1–3 mg / g, and optionally an oleic acid (C18:1) content of less than 5 mg / g, preferably up to 2 mg / g, based on the total dry weight of the cultured avocado cells.

[0088] In certain preferred embodiments, avocado cells are cultured in cell suspension culture for a sufficient time and under conditions to result in a polyunsaturated fatty acid ratio of 30–70% by weight, preferably 40–65% by weight, more preferably 50–60% by weight, and optionally 5–15% by weight, preferably 8–12% by weight, of monounsaturated fatty acids, and / or a saturated fatty acid ratio of 20–40% by weight, preferably 25–40% by weight, more preferably 30–35% by weight, based on the total weight of lipids in the cultured avocado cells. If desired, avocado cells are cultured in cell suspension culture for a sufficient time and under conditions to result in a proportion of 30–55% by weight, preferably 35–50% by weight, more preferably 40–50% by weight of linoleic acid (C18:2n-6), and / or 5–15% by weight or 6–11% by weight of α-linoleic acid (C18:3n-3), and optionally 5–10% by weight of oleic acid (C18:1), and optionally 20–30% by weight or 20–25% by weight of palmitic acid (C16:0), based on the total weight of lipids in the cultured avocado cells.

[0089] In certain preferred embodiments, avocado cells are cultured in cell suspension culture for a sufficient time and under conditions to result in a sterol content of 0.5–2.0 mg / g, preferably 0.5–1.5 mg / g, more preferably 0.75–1.25 mg / g, and optionally 0.5–1.0 mg / g of β-sitosterol, based on the total dry weight of the cultured avocado cells.

[0090] In certain preferred embodiments, the culture time and conditions of the cell suspension culture are selected to result in cultured avocado cells that are substantially (naturally) free from one or more or all of 2-pentadecylfuran, 2-heptadecadienylfuran, 3-hexen-1-ol, caryophyllene oxide, trans-α-bergamotene, caryophyllene, and / or tocopherol, preferably at least free from caryophyllene and / or tocopherol, more preferably free from at least both.

[0091] In certain preferred embodiments, the conditions for cell suspension culture are: Temperatures within the range of 22°C to 28°C, preferably 24°C to 26°C. Induction using one or more of the following: preferably methyl jasmonate at a concentration of 20-500 μM, preferably jasmonic acid at a concentration of 20-500 μM, preferably ethylene at a concentration of 50-200 mg / l, more preferably about 100 mg / l, preferably etephon at a concentration of 50-200 mg / l, more preferably about 100 mg / l, salicylic acid, preferably chitosan at a concentration of 50-500 mg / l, more preferably about 200 mg / l, Zn ions, Cu ions, Fe ions, and / or Mn ions, and / or One or more of the following illumination regimes are selected: dark / light, 8 hours / 16 hours dark / light, or complete darkness (no light period). Such culture conditions may provide the cultured avocado cells with the desired amount of the desired compound. Such induction may increase the levels of (one or more) flavor compounds (volatile substances), color, and / or (one or more) phenols in the cultured avocado cells.

[0092] Culturing avocado cells in a cell suspension culture for a sufficient amount of time and under conditions to result in a specific content of one or more specific compounds may be a step in controlling the production of the above compounds in the cell suspension culture.

[0093] Preferably, the culture time in cell suspension culture is within the range of 4 to 20 days, preferably 7 to 15 days, more preferably 7 to 12 days or 7 to 11 days. It was found that the duration of cell culture in suspension culture can affect the content of MUFA, PUFA, SAFA, and sterols in the recovered or harvested cultured avocado cells. As shown in this example, particularly high PUFA content was observed when the culture period in cell suspension culture was within the range of 7 to 11 days. The amount of SAFA in cultured avocado cells appeared to follow a similar trend to PUFA, but the MUFA content was observed to be higher from day 4 to 7, after which the MUFA content began to decrease. Increasing the culture time in cell suspension culture increased the amount of sterols in cultured avocado cells.

[0094] The liquid culture medium for cell suspensions may contain growth regulators. Preferably, the liquid medium contains at least one cytokinin or cytokinin-like plant growth regulator (CN) and at least one auxin (AUX). Examples of suitable CNs include kinetin, 6-(γ,γ-dimethylallylamino)purine, zeatin, or any combination thereof, and examples of suitable AUXs include α-naphthaleneacetic acid, indoleacetic acid, picoram, or any combination thereof, preferably α-naphthaleneacetic acid, indoleacetic acid, or any combination thereof. Preferably, the CN concentration is 0.1 to 5.0 ppm and the AUX concentration is 1.0 to 10 ppm.

[0095] The liquid medium may be Murashige and Skoog medium (MS medium as described in Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15, 473-497), which optionally contains growth regulators and / or Glu and Gly as described above.

[0096] Preferably, cell suspension culture is carried out in an (ambient) air atmosphere.

[0097] In certain preferred embodiments, the liquid culture medium is At least one CN selected from kinetin, 6-(Y,y-dimethylallylamino)purine, zeatin, or any combination thereof, wherein the CN concentration in the liquid medium is preferably 0.1 to 5.0 ppm, and at least one AUX selected from α-naphthaleneacetic acid, indoleacetic acid, or any combination thereof, wherein the AUX concentration in the liquid medium is preferably 1.0 to 10 ppm, The product comprises glycine (Gly) and glutamic acid (Glu), preferably in a total amount of 1 to 15 mM, more preferably in a total amount of 0.01 to 0.05 mM Gly and 0.5 to 1.5 mM Glu.

[0098] In certain preferred embodiments, cell suspension culture is preferably performed on an orbital shaker at 90-120 rpm, preferably about 110 rpm, at a temperature in the range of 22-28°C, such as about 23°C, using growth regulators, preferably at least one cytokinin or cytokinin-like plant growth regulator (CN) and at least one auxin (AUX), preferably in an amount of 0.1-5.0 ppm, more preferably about 0.1 ppm, preferably kinetin, and preferably in an amount of 1.0-10 ppm, preferably about 1.0 ppm, of α-naphthaleneacetic acid (NAA), using Murashige and Skoog medium (Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15). Cell suspension culture is initiated with 0.03 to 0.07 g of callus per gram of liquid medium (MS medium as described in 473-497), preferably carried out under an (ambient) air atmosphere, and optionally the liquid medium contains 0.01 to 0.05 mM Gly and 0.5 to 1.5 mM Glu.

[0099] Suspension cultures can be subcultured every 7 to 16 days, for example, every 10 days. For subculture, the biomass can be diluted with a biomass:liquid ratio of approximately 1:3 to 1:5 wt:wt.

[0100] Preferably, the callus and cultured avocado cells consist substantially of undifferentiated cells.

[0101] The method of the present invention may include a step of analyzing avocado cells in a cell suspension culture, which includes a step of obtaining avocado cells from the cell suspension culture, preferably drying the obtained avocado cells preferably by freeze-drying, and preferably analyzing the dried avocado cells to determine the content of (one or more) specific compounds. Examples of such compounds may be major nutrients, preferably at least proteins, more preferably at least proteins and lipids, polyunsaturated fatty acids, linoleic acid and / or alpha-linoleic acid, and / or any other (one or more) compounds discussed above. Following the analysis step, a step of deciding whether to continue culturing or harvest the avocado cells based on the determined content of (one or more) specific compounds is followed. Such an analysis step, and preferably a subsequent determination step, is a means of controlling the content of (one or more) specific compounds in cultured avocado cells.

[0102] Avocado cells can be harvested from cell suspension culture using any suitable harvesting method or technique. For example, such a method may include the step of harvesting avocado cells from cell suspension culture by vacuum filtration. Preferably, the harvested cells are washed to remove the culture medium.

[0103] If desired, harvested and washed cultured avocado cells may be frozen and stored, if desired, in the range of -90°C to -15°C, preferably in the range of -80°C to -18°C, or cultured avocado cells may be stored in liquid nitrogen (liquid N2) at a temperature below -130°C, preferably below -140°C. Typically, the frozen cells are thawed and, if desired, powdered and / or dried before being used as a food or in food.

[0104] If desired, the obtained cultured avocado cells are subjected to lysis in a liquid-to-cultured avocado cell ratio of preferably 0.7:1 to 3:1 wt / wt (wet weight), more preferably about 1:1 wt / wt (wet weight), to obtain cell lysates. The liquid may be water, such as sterile Milli-Q water, or an aqueous solution with appropriate additions, for example. In other words, cultured avocado cells may be in the form of cell lysates. The cultured avocado cells subjected to lysis are cryopreserved as described above and may be thawed before lysis, for example, in an ice bath. The cultured avocado cells subjected to lysis are typically provided without drying the cells. That is, the cultured avocado cells subjected to lysis are typically not dried (freeze-dried). Providing the cultured avocado cells of the present invention as a cell lysate may have a positive effect on the mouthfeel of the cells, for example, the mouthfeel of the cells may be perceived as less sandy, and / or providing the cultured avocado cells of the present invention as a cell lysate may have an effect that facilitates processing of the cultured avocado cells, either alone or together with other food ingredients to form food products.

[0105] Cell lysates may be cryopreserved and, if desired, stored at a temperature in the range of -90°C to -15°C, preferably in the range of -80°C to -18°C, or the cell lysates may be stored in liquid nitrogen (liquid N2) at a temperature below -130°C, preferably below -140°C. Frozen cell lysates may be thawed before use as a food or in food. In certain preferred embodiments, the method includes the step of powdering cultured avocado cells, which preferably includes drying the cultured avocado cells preferably by freeze-drying and / or spray-drying, more preferably by freeze-drying, followed optionally by mechanical powdering such as homogenization, grinding, and / or crushing. Freeze-drying may result in a powder that is particularly palatable or pleasant. In certain embodiments, cultured avocado cells are subjected to lysis and then to freeze-drying. Such cells may become palatable.

[0106] The cultured avocado cells of the present invention can be powdered. Typically, the powdering includes the steps of drying the cultured avocado cells and, optionally, subjecting the dried cells to mechanical powdering or homogenization.

[0107] Preferably, the step of pulverizing cultured avocado cells includes freeze-drying and / or spray-drying, followed optionally by mechanical pulverization or homogenization. Cultured avocado cells may be sufficiently pulverized by simply drying, particularly by freeze-drying. However, additional pulverization or homogenization of the powder may be utilized if necessary or desired.

[0108] Mechanical pulverization may include homogenization, grinding, and / or crushing.

[0109] Preferably, the method of the present invention includes the step of using cultured avocado cells in or as food for human consumption. Any chemicals and solvents used in the method of the present invention are preferably selected so as to yield cultured avocado cells suitable for use in food for human consumption. As used herein, “suitable for use in food for human consumption” does not necessarily imply regulatory approval.

[0110] This specification further provides a food comprising cultured avocado cells as described herein. Preferably, the food comprises 0.1 to 100% by weight, more preferably 0.5 to 90% by weight, more preferably 1.0 to 80% by weight, even more preferably 5 to 70% by weight, and most preferably 10 to 65% by weight of the cultured avocado cells of the present invention, based on the total weight of the food, preferably by any one of the embodiments described above or by any one of the embodiments of this disclosure.

[0111] The cultured avocado cells of the present invention may be used in or as a food, and the content of avocado cells in the food may preferably be as defined above. The cultured avocado cells may be used in food obtained from cell suspension culture after harvesting (for example, by freezing followed by thawing for preservation purposes, or without them) and optionally after washing, and / or the cultured avocado cells may be provided in food as a lysate and / or preferably as a powder obtained as described above. Preferably, the cultured avocado cells may be provided in food as a powder, more preferably as a freeze-dried or lyophilized powder, or included in food.

[0112] Foods can be nutritional supplements such as protein powder, spreads, dips, guacamole, flakes, dumplings, squeezed biomass for sushi, muesli, granola, or smoothies including smoothie bowls.

[0113] The mesocarp of a mature, fresh avocado fruit can be described as having a smooth, buttery and / or creamy texture and a rich, nutty flavor. The mesocarp of an unripe avocado fruit can be described as having a watery, firm texture and a greasy taste.

[0114] The cultured avocado cells of the present invention do not have the shape and / or texture of an avocado fruit or its mature mesocarp itself. However, the cultured avocado cells can be formulated to obtain a texture at least similar to mashed mature avocado mesocarp. Such formulations may include structural components such as thickeners and optionally (added) water, especially when the cultured avocado cells are provided in formulation as a powder. These formulations can be used, for example, in guacamole, (one or more) smoothies, (one or more) spreads, and / or (one or more) dips. Thus, the food products of the present invention may contain one or more food-grade additives, preferably (one or more) thickeners, (one or more) antioxidants, (one or more) colorants, and / or (one or more) flavorings. Furthermore, fresh cultured avocado cells as described herein have been found to have a pleasant flavor and aroma, including avocado flavor characteristics.

[0115] In certain embodiments, the food comprises, based on the total weight of the food, 1 to 5% by weight of a thickener and / or 1 to 4% by weight of an antioxidant.

[0116] To provide and / or maintain a green or greenish color and / or yellow or yellowish color similar to that of a mature avocado mesocarp, (one or more) colorants and / or (one or more) antioxidants may be applied to the formulation containing the cultured avocado cells of the present invention. For example, citric acid may be used to bleach brown and / or gray discoloration of the cultured avocado cells and / or to prevent their formation. For example, aqueous colorant solutions may be provided from kale leaves and / or spinach leaves, for example, by immersing chopped leaves in water and then filtering them.

[0117] Preferably, cultured avocado cells are used in or as a food in powder form. Providing cultured avocado cells as a powder makes it possible to use them in existing processes and / or existing equipment for avocado fruit powder. For example, some commercially available guacamole is made using powdered avocado fruit mesocarp. In such processes and / or powdered products, cultured avocado cells can replace all or part of avocado fruit powder without investment in and / or modification of processes and / or equipment.

[0118] The embodiments and implementations of the present invention are further discussed in the following numbered sections. 1. Cultured avocado cells, comprising at least 15% by weight of protein based on the total dry weight of the cultured avocado cells. 2. Based on the total dry weight of cultured avocado cells, 15-30% by weight, preferably 20-30% by weight, more preferably 25-30% by weight of protein, and / or Cultured avocado cells according to item 1, comprising 0.5 to 10% by weight, preferably 1 to 7% by weight, more preferably 1.5 to 6% by weight, and even more preferably 2 to 5% by weight of lipids. 3. Cultured avocado cells according to item 1 or 2, wherein the cultured avocado cells contain at least 5 mg / g, preferably 5-25 mg / g, more preferably 7-20 mg / g, more preferably 10-18 mg / g, and even more preferably 10-15 mg / g of polyunsaturated fatty acids based on the total dry weight of the cultured avocado cells, and optionally, based on the total dry weight of the cultured avocado cells, the linoleic acid (C18:2n6) content is at least 5 mg / g, for example 5-20 mg / g, preferably at least 8 mg / g, more preferably 8-15 mg / g, and even more preferably 8-10 mg / g, and / or the alpha-linoleic acid (C18:3n3) content is 1 mg / g or more, preferably 1.50 mg / g or more, more preferably 1-3 mg / g, and even more preferably 1.5-2 mg / g. 4. Cultured avocado cells according to any one of claims 1 to 3, wherein the cultured avocado cells contain 3 to 15 mg / g, preferably 5 to 10 mg / g, saturated fatty acids and / or less than 10 mg / g, preferably less than 5 mg / g, more preferably 1 to 3 mg / g, monounsaturated fatty acids based on the total dry weight of the cultured avocado cells, and optionally, the oleic acid (C18:1) content in the cultured avocado cells is less than 5 mg / g, preferably up to 2 mg / g, based on the total dry weight of the cultured avocado cells. 5. Cultured avocado cells according to any one of claims 1 to 4, comprising 10 to 65% by weight, preferably 14 to 65% by weight, more preferably 25 to 64% by weight, and even more preferably 55 to 64% by weight of carbohydrates, based on the total dry weight of the cultured avocado cells. 6. Cultured avocado cells according to any one of claims 1 to 5, wherein the cultured avocado cells contain 0.5 to 2.0 mg / g, preferably 0.5 to 1.5 mg / g, more preferably 0.75 to 1.25 mg / g of sterols based on the total dry weight of the cultured avocado cells, and optionally, the β-sitosterol content in the cultured avocado cells is 0.5 to 1.0 mg / g based on the total dry weight of the cultured avocado cells. 7. Cultured avocado cells are substantially free of caryophyllene and / or tocopherol, and / or Cultured avocado cells according to any one of claims 1 to 6, wherein the cultured avocado cells contain (one or more) catechins and / or (one or more) procyanidins, preferably (one or more) catechins and / or (one or more) oligomeric procyanidins. 8. Cultured avocado cells according to any one of claims 1 to 7, wherein the cultured avocado cells are in the form of a cell lysate or powder, preferably a powder obtained by freeze-drying and / or spray-drying, more preferably freeze-drying, followed optionally by mechanical powdering such as homogenization, grinding and / or crushing. 9. Cultured avocado cells as described in any one of items 1 to 8, wherein the cultured avocado cells are food-grade avocado cells intended for human consumption. 10. A method for producing cultured avocado cells, The process of providing avocado cells, The process involves initiating cell suspension culture by seeding the provided avocado cells into a liquid medium, and culturing the avocado cells in cell suspension culture for a sufficient time and under conditions to result in a protein content of at least 15% by weight based on the total dry weight of the avocado cells. A method comprising the step of collecting avocado cells from a cell suspension culture to obtain cultured avocado cells. 11. The process of providing avocado cells is a process of growing callus from avocado on a solid growth medium, The method according to item 10, comprising the step of providing avocado cells from callus. 12. The step of providing avocado cells is a step of selecting avocado cells that produce polyunsaturated fatty acids in cell suspension culture, wherein the avocado cells are preferably selected from callus. The process includes providing selected avocado cells, In cell suspension culture, the culture process is The method according to claim 10 or 11, comprising the step of culturing cultured avocado cells for a sufficient time and under conditions to result in a polyunsaturated fatty acid content of at least 5 mg / g based on the total dry weight of the cultured avocado cells. 13. The method according to any one of claims 10 to 12, wherein the culture time and conditions of the cell suspension culture are selected to result in a lipid content of 0.5 to 10% by weight, preferably 1 to 7% by weight, more preferably 1.5 to 6% by weight, and even more preferably 2 to 5% by weight, based on the total dry weight of the cultured avocado cells, and / or substantially free of caryophyllene and / or tocopherol. 14. The method according to any one of claims 10 to 13, wherein the culture time in cell suspension culture is in the range of 4 to 20 days, preferably 7 to 15 days, more preferably 7 to 12 days or 7 to 11 days. 15. The conditions for cell suspension culture are, Temperatures within the range of 22°C to 28°C, preferably 24°C to 26°C. Induction using one or more of the following: preferably methyl jasmonate at a concentration of 20-500 pM, preferably jasmonic acid at a concentration of 20-500 pM, preferably ethylene at a concentration of 50-200 mg / l, more preferably about 100 mg / l, preferably etephon, salicylic acid at a concentration of 50-200 mg / l, more preferably about 100 mg / l, and / or chitosan at a concentration of 50-500 mg / l, more preferably about 200 mg / l, Zn ions, Cu ions, Fe ions, and / or Mn ions, and / or The method described in any one of items 10-14, selected from one or more of the following: a dark / light illumination regime, 8 hours / 16 hours of dark / light, or complete darkness (no light period). 16. The method according to any one of items 10 to 15, comprising the step of using cultured avocado cells in or as a food for human consumption. 17. The method according to any one of claims 10 to 16, wherein the method comprises a step of powdering cultured avocado cells, wherein the step preferably comprises drying the cultured avocado cells preferably by freeze-drying and / or spray-drying, more preferably by freeze-drying, and subsequently, optionally, mechanical powdering such as homogenization, grinding and / or crushing. 18. The method according to any one of items 10 to 17, wherein the callus and cultured avocado cells consist substantially of undifferentiated cells. 19. The method according to any one of claims 10 to 18, wherein the callus grows from avocado fruit material or avocado plant section, preferably a section or multiple sections from an avocado seed, and the seed is optionally subjected to germination before cutting. 20. The method according to any one of items 10 to 19, further comprising the step of sterilizing the part of the avocado from which the callus will be grown before allowing the callus to grow. 21. Food, comprising, based on the total weight of the food, 0.1 to 100% by weight, preferably 0.5 to 90% by weight, more preferably 1.0 to 80% by weight, more preferably 5 to 70% by weight, and even more preferably 10 to 65% by weight of avocado culture cells as described in any one of items 1 to 9. 22. The food according to claim 21, comprising one or more food-grade additives, preferably one or more of (one or more) thickeners, (one or more) antioxidants, (one or more) colorants, and / or (one or more) flavorings. 23. The food according to item 21 or 22, comprising (one or more) 1 to 5% by weight of a thickener and / or (one or more) 1 to 4% by weight of an antioxidant, based on the total weight of the food. 24. Foods described in any one of sections 21-23, which are dietary supplements, spreads, dips, guacamole, flakes, dumplings, squeezed biomass for sushi, muesli, granola, or smoothies. [Examples]

[0119] Example 1 - Callus culture To establish callus from avocado fruit, sterilized fruit material or sections of obtained in vitro sterile plants were incubated with sucrose (3% w / v), the plant growth regulator kinetin (0.1 ppm), and α-naphthaleneacetic acid (NAA) (1.0 ppm) on Murashige and Skoog medium (MS medium as described in Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15, 473-497), and then solidified on agar (6.5 g / l). The incubation temperature for the samples was 22 ± 2°C, but samples were typically incubated in both dark and light conditions. Illumination was provided with a day:night photoperiod (16:8 hours) and 30-60 μmol / m³. 2 Normal light with s irradiation was used. The resulting soft callus, which typically forms within 7-21 days, was transplanted into fresh culture medium.

[0120] The above-mentioned MS medium and lighting were used for maintaining and culturing the obtained callus, and the temperature used was 24°C. The callus cultures were subcultured every 3 to 5 weeks.

[0121] Example 1.1 Origin of the avocado fruit: Chile, ripe avocado fruit purchased at a local market in Finland. Seeds from mature avocado fruits were germinated in water for two months, surface-sterilized, and cut into small pieces. The pieces were incubated on the solid MS medium described above. The resulting callus cultures were separated into two lines, maintained in light (Example 1.1V) and dark (Example 1.1P). The main color of both callus lines was light gray, although occasionally the callus in Example 1.1V could partially turn pink.

[0122] Example 1.2 Origin of the avocado fruit: Spain. I purchased a ripe avocado fruit at a local market in Finland. Seeds from mature avocado fruits were sterilized and sown in sterile MS medium that did not contain plant growth regulators to support in vitro plant growth. Sections of the sterile stems of the resulting in vitro plants were incubated on MS medium containing the aforementioned plant growth regulators to establish callus. This callus was maintained in light, and after 3–6 weeks of incubation, some of the light brown callus changed to a pinkish color.

[0123] Example 1.3 Origin of the avocado fruit: Chile, Reed variety. I purchased a mature avocado fruit at a local market in Finland. Material from the inner layer of avocado fruit, between the pericarp and mesocarp (flesh), was used to establish callus. The resulting callus lineage was maintained in light, and after several weeks of incubation, the color of the callus, which was initially brownish, changed to a partially pinkish color.

[0124] Example 1.4 Origin of the avocado fruit: Spain, Hass variety. I purchased a ripe avocado fruit at a local market in Finland. Seeds of mature avocado fruits were surface-sterilized, and the embryos were removed from the seeds and used to establish callus culture. The resulting callus cultures were maintained as two lines, cultured in light (Example 1.4V) and in darkness (Example 1.4P). The color of the callus line in Example 1.4P was light gray (almost white), but after several weeks of incubation in light, the callus line in Example 1.4P changed to a pale pink color.

[0125] Example 2 - Cell Suspension Culture Suspension culture was initiated at 23°C on an orbital shaker at 110 rpm under atmospheric conditions using 0.5–1 g of well-growing callus obtained in the same manner as above in 15 mL of MS medium containing kinetin (0.1 ppm) and NAA (1.0 ppm) in a 50 mL Erlenmeyer flask. The culture was regularly subcultured every 10 days and gradually scaled up to a 250 mL flask containing 60 mL of culture. For subculture, the biomass was diluted in a 1:3 wt:wt biomass:liquid medium. Culture was carried out under an 8-hour / 16-hour dark / light illumination regime or in the dark.

[0126] During the culture of the suspension culture, the fatty acid and sterol content, specifically saturated fatty acids (SAFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and sterols, was analyzed according to the culture time. The results are shown in Table 1 and Figure 1. The fatty acid content was determined according to the method reported by Glaser et al. 2010 High-Throughput Analysis of Total Plasma Fatty Acid Composition with Direct In Situ Transesterification, PLOS ONE (https: / / doi.org / 10.1371 / journal.pone.0012045), and the sterol content was determined according to the method described by Seppanen-Laakso et al. 2017 UPLC-ELSD Analysis of Algal Lipid Classes and Derivatization of Bound and Free Fatty Acids and Sterols for GC-MS Methods, Biofuels from Algae, vol. 1980, pp. 223-232 (DOI: 10.1007 / 7651_2017_109).

[0127] TIFF2026521485000001.tif48164

[0128] As can be seen from Table 1 and Figure 1, PUFA content increased during the first week of culture, with the highest PUFA content measured at culture time 7–11 days, after which PUFA content began to decrease. SAFA showed a similar trend, but the change in SAFA content over culture time was not as pronounced compared to PUFA. MUFA content peaked at culture time 4–7 days, after which it began to decrease. The change in MUFA content over culture time was smaller compared to PUFA and SAFA. Sterol content increased over culture time 2–23 days, with the highest sterol content measured at 23 days.

[0129] As can be seen from Table 1 and Figure 1, at all measurement points from 2d to 23d, the PUFA content was much higher than that of MUFA and SAFA, respectively.

[0130] Avocado cells were harvested from cell suspension culture by vacuum filtration.

[0131] Example 3 - Analysis of cultured avocado cells Example 3.1 Analysis of Major Nutrients The cultured avocado cells according to this disclosure were obtained from a cell suspension culture initiated from callus grown in the same manner as described in Example 2 and as described in Example 1. The culture time in the cell suspension culture was 11 days.

[0132] Major nutrient and energy content were measured in cultured avocado cells and compared with values ​​reported for fresh avocado fruit. Results and analytical methods are reported in Table 1. Neither the cultured avocado cells nor the avocado fruit were dried before analysis. The water content of the cultured avocado cells is reported in Table 1.

[0133] TIFF2026521485000002.tif130164

[0134] As can be seen directly from Table 1, the cultured avocado cells in this example have a much lower lipid content and a significantly higher carbohydrate content than fresh avocado fruit. Taking moisture into account, we can conclude that the protein content of cultured avocado cells is very high, at approximately 28% by weight based on the total dry weight of the avocado cells. This is clearly higher than that of fresh avocado fruit. For example, assuming a moisture content of approximately two-thirds (w:w), the protein content of fresh avocado fruit is only about 8% by weight based on the total dry weight of the fresh avocado fruit. Therefore, the protein content of cultured avocado cells in this example was more than three times, approximately 3.5 times, compared to the protein content of fresh avocado fruit based on their respective dry weights.

[0135] Example 3.2 Fatty Acid Analysis Two samples of cultured avocado cells according to this disclosure, C1 and C2, were obtained from cell suspension cultures initiated from callus grown in the same manner as described in Example 2 and as in Example 1. The culture time in the cell suspension cultures was 11 days. Prior to analysis, the cultured avocado cells were freeze-dried, and the freeze-dried cells were used for analysis.

[0136] The fatty acid content was determined according to the method reported by Glaser et al. 2010 High-Throughput Analysis of Total Plasma Fatty Acid Composition with Direct In Situ Transesterification, PLOS ONE (https: / / doi.org / 10.1371 / journal.pone.0012045), and the FAME, FFA, and sterol content was determined according to the method described by Seppanen-Laakso et al. 2017 UPLC-ELSD Analysis of Algal Lipid Classes and Derivatization of Bound and Free Fatty Acids and Sterols for GC-MS Methods, Biofuels from Algae, vol. 1980, pp. 223-232 (DOI: 10.1007 / 7651_2017_109).

[0137] For reference, two samples of mesocarp from mature avocado fruit, F1 and F2, were used. Both F1 and F2 were freeze-dried before analysis, and the freeze-dried samples were analyzed.

[0138] The results and calculated average values ​​are reported in Tables 2-4.

[0139] TIFF2026521485000003.tif206164

[0140] TIFF2026521485000004.tif208164

[0141] As can be seen from Tables 2 and 3 and Figure 2, the fatty acid profiles of C1 and C2 differ from those of F1 and F2. For example, C1 and C2 contained linoleic acid (and alpha-linoleic acid) as their (one or more) main fatty acids (FAs), while F1 and F2 contained oleic acid as their main FA. As can be seen from Table 3 and Figure 2, the proportion of PUFAs in total lipids was much higher in C1 and C2 than in F1 and F2, while the proportion of MUFAs was lower in C1 and C2 compared to F1 and F2, and the proportion of SAFAs was higher. PUFAs are generally considered particularly desirable FAs because they can provide health benefits. While SAFAs may be considered unhealthier compared to unsaturated fatty acids because their total lipid content is much lower in C1 and C2, as seen in Table 2, the SAFA content, expressed as mg / g based on the total dry weight of each sample, was actually much lower in C1 and C2 compared to F1 and F2, by more than three or even four times.

[0142] In addition to sterols measured according to Seppanen-Laakso et al. 2017 UPLC-ELSD Analysis of Algal Lipid Classes and Derivatization of Bound and Free Fatty Acids and Sterols for GC-MS Methods, Biofuels from Algae, vol. 1980, pp. 223-232 (DOI: 10.1007 / 7651_2017_109), the contents of tyramine, 2-penadecylfuran, and 2-heptadecadienylfuran are reported in Table 4 and Figure 3.

[0143] TIFF2026521485000005.tif109164

[0144] As can be seen from Table 4 and Figure 3, C1 and C2 contained more sterols than F1 and F2, but based on their respective dry weights, the sterol content was more than four times, and even more than five times, higher. In particular, the β-sitosterol content was higher in C1 and C2 compared to F1 and F2, based on their respective dry weights.

[0145] Example 3.3 Analysis of volatile substances Two samples of cultured avocado cells according to this disclosure, C1-V and C1-P, were obtained from cell suspension cultures starting from callus grown in the same manner as described in Example 2, and in the same manner as in Example 1.1V (C1-V) and Example 1.1P (C1-P). In the suspension cultures, C1-V was cultured under an 8-hour / 16-hour dark / light illumination regime, and C1-P was cultured in the dark.

[0146] Prior to analysis, cultured avocado cells were freeze-dried, and the analysis was performed using the freeze-dried cells. The analytical method is described in Example 3.0 below.

[0147] For reference, three samples of mesocarp from mature avocado fruit, F1, F2, and F3, were used. F1, F2, and F3 were freeze-dried before analysis, and the freeze-dried samples were analyzed as described in Example 3.0.

[0148] The results are reported in Tables 5-12 and Figures 4-12.

[0149] TIFF2026521485000006.tif64157

[0150] As can be seen from Table 5 and Figure 4, C1-P and C1-V contained significantly more benzaldehyde than F1-F3. C1-V and C1-P contained slightly more trans-2-nonenal than F1-F3, and significantly less 1-octenal.

[0151] TIFF2026521485000007.tif63164

[0152] As can be seen from Table 6 and Figure 5, C1-V and C1-P contained significantly less 1-hexanol than F1-F3. The content of tetradecane and 3-octen-2-one was also somewhat lower in C1-V and C1-P than in F1-F3, respectively.

[0153] TIFF2026521485000008.tif63164

[0154] As can be seen in Table 7 and Figure 6, C1-V and C1-P contained more 2-octenal and 1-octen-3-ol, respectively, than F1-F3, and contained less furfural and far less copaene.

[0155] TIFF2026521485000009.tif23661

[0156] As seen in Table 8 and Figure 7, C1-V and C1-P contained significantly more hexanal and far less dodecane than F1-F3. The 2-hexenal and 1-pentanol content in C1-V, C1-P, and F1-F3 was relatively similar, but F1-F3 contained more 2-heptenal and less 2-pentylfuran than C1-V and C1-P.

[0157] TIFF2026521485000010.tif62164

[0158] As seen in Table 9 and Figure 8, acetaldehyde content was significantly lower in C1-V and C1-P than in F1-F3, methanol content was at somewhat similar levels, but ethanol and pentanal content were higher in C1-V and C1-P than in F1-F3, respectively. Ethanol content was particularly high in C1-V, which may suggest that the light-dark regime influences ethanol content.

[0159] TIFF2026521485000011.tif59164

[0160] As shown in Table 10 and Figure 9, the content of isopropyl myristate and octanoic acid was much higher in C1-V and C1-P than in F1-F3, respectively. The content of isopropyl myristate was not reported for F2 and F3. C1-V, C1-P, and F1-F3 contained fairly similar, seemingly small amounts of 2-methoxy-4-vinylphenol.

[0161] TIFF2026521485000012.tif63164

[0162] As shown in Table 11 and Figure 10, C1-V and C1-P contained significantly more isopropyl palmitate and 2,4-di-tert-butylphenol, respectively, than F1-F3. The diethyl phthalate content was at fairly similar levels in the samples C1-V, C1-P, and F1-F3 that were examined.

[0163] TIFF2026521485000013.tif72164

[0164] As seen in Table 12 and Figure 11, the content of 2-octenal-2-butyl was significantly higher in C1-V and C1-P compared to F1-F3, where the content of 2-octenal-2-butyl was not reported. Neither C1-V nor C1-P had reported caryophyllene oxide content. However, caryophyllene oxide was present in F1-F3 in clearly significant amounts. C1-V and C1-P contained significantly more benzyl alcohol compared to F1-F3, where only very small amounts were reported. C1-V and C1-P had slightly higher hexanoic acid content than F1-F3, and the content of 2,4-decadienal was somewhat similar among the C1-V, C1-P, and F1-F3 samples examined.

[0165] Figure 12 shows the analyzed trans-α-bergamotene and caryophyllene content of freeze-dried cultured avocado cells (C1-V and C1-P) and freeze-dried avocado fruit mesocarp (F1-F3). As seen in Figure 12, C1-V and C1-P had virtually no trans-α-bergamotene and caryophyllene content, while F1-F3 appeared to have considerably higher content.

[0166] Example 3.0 Method for analyzing volatile substances Organic compounds (volatile substances) were identified from freeze-dried cultured avocado cells and freeze-dried mesocarp of mature avocado fruit according to this disclosure.

[0167] For the analysis of volatile compounds, 100 mg of sample material was accurately weighed and placed in a preheated (70°C, 1 hour) headspace vial. Milli-Q water (1 ml) was added, and the vial was sealed with a preheated magnetic cork. GC-MS analysis was performed using an Agilent HP-Innowax 19091N-136I (60 m × 250 μm × 0.25 μm) column, an Agilent 7890B oven, and a 5977B mass chromatograph equipped with a CTC / PAL autosampler. The SPME (solid-phase microextraction) fiber used was 1 cm Supelco 57329-U DVB / CAR / PDMS. The sample was incubated at 80°C (1 min), and then the fiber was continuously agitated in the headspace vial for 30 minutes (250 rpm, 20 seconds on, 10 seconds off). The fiber was desorbed at the inlet of the GC oven for 6 minutes. The fibers were adjusted between samples (10 minutes, 270°C). Analysis was performed in splitless mode (250°C) at a flow rate of 1.2 ml / ml. The temperature program used was 40°C for 3 minutes, then 10°C / min up to 240°C, which was maintained for 7 minutes. MS detection was performed in the range of 25–600 amu.

[0168] Example 3.1 - Analysis of catechins and procyanidins The cultured avocado cell samples C1-V, C1-P1, C1-P2, C2-V, and C3-V according to this disclosure were obtained from cell suspension cultures initiated from callus grown in Example 1.1V (C1-V), Example 1.1P (C1-P1, C1-P2), Example 1.2 (C2-V), and Example 1.4V (C3-V), respectively, by carrying out the same procedures as described in Example 2.

[0169] Qualitative analysis of catechins and procyanidins was performed using ULC-DAD-Qtof-MS on a 10 mg methanol extract sample, and the compound (polyphenol) was identified according to Rosero et al., Analysis of Phenolic Composition of Byproducts (Seeds and Peels) of Avocado (Persea americana Mill.) Cultivated in Colombia, Molecules 2019, 24(17), 3209; (https: / / doi.org / 10.3390 / molecules24173209). The results are shown in Figure 13.

[0170] As shown in Figure 13, all of the samples examined—C1-V, C1-P1, C1-P2, C2-V, and C3-V—showed at least several catechin and procyanidin peaks, suggesting their presence. The largest peak was obtained in C2-V and C3-V, particularly in C2-V. Therefore, it appears that several (one or more) catechins and / or procyanidins, and even oligomeric procyanidins, are present in C1-V, C1-P1, C1-P2, C2-V, and C3-V, and at least in C2-V and C3-V.

[0171] Example 4 - Food containing cultured avocado cells The guacamole-type food according to this disclosure was formed using cultured avocado cells that were frozen and thawed before use.

[0172] Cultured avocado cells were obtained from suspension culture starting from callus that had grown in the same manner as described in Example 2 and as in Example 1.

[0173] Cultured avocado cells were prepared as described below.

[0174] Food-grade thickeners, namely 0.4% by weight of psyllium (Virtasalmen Viljat) and 2% by weight of xanthan gum (Farina; a solution made from Puhdistamo's MCT oil), were added to cultured avocado cells and mixed to provide a mixture with a desired structure similar to the mesocarp of mashed avocado fruit.

[0175] The mixture contained 2.5% by weight of citric acid as an antioxidant and to bleach the brownish color.

[0176] The above weight percentages are based on the total weight of the mixture.

[0177] Subsequently, aqueous kale leaf extract was mixed with the mixture as a coloring agent at a rate of 30 ml per 100 g of avocado cells. The kale leaf extract was obtained as follows: 30 g of kale leaves (in 70 ml of water, at room temperature) were crushed using a Pamix for approximately 1 minute, then centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected as the kale leaf extract.

[0178] Subsequently, the above preparation containing cultured avocado cells was mixed with chili peppers, onions, tomatoes, and coriander to obtain guacamole.

[0179] Figure 14 shows a photograph of guacamole formed using a preparation containing (complete) cultured avocado cells. As can be seen in Figure 14, the guacamole containing cultured avocado cells is very similar in appearance and texture to conventional guacamole made from the mesocarp of avocado fruit.

[0180] Example 5 - Cell suspension culture using amino acid supply Cultured avocado cells were obtained from a suspension culture starting from callus grown in the same manner as in Example 1, but with the following modifications: the avocado cell culture was propagated in a flask (250 ml Erlenmeyer, 70 ml working volume), and the mixed suspension was divided into a 100 ml Erlenmeyer flask (20 ml working volume) on day 5. Glycine and glutamic acid were dissolved in water, filtered, and added to the cell suspension cultures at final concentrations of 0.03 mM and 1 mM, respectively. They were not added to the control culture. After supply, the culture was continued as usual from the start to day 10. Cells were collected, biomass was measured, and the cells were freeze-dried before protein analysis.

[0181] Protein analysis (total amino acid composition and content) was performed as follows. Reagents and materials: AccQ-Tag reagent kit, Mass TRAK™ amino acid analysis concentrate A, and eluate B were obtained from Waters (Milford, MA, USA). Amino acid standard solutions, physiological amino acid standards (basic), L-isoleucine, glutamine, and norvaline were obtained from Sigma-Aldrich (St. Luis, Missouri, USA).

[0182] To determine the total amino acid content, a sample (5 mg, accurately weighed) was first oxidized with a fresh formic acid solution (0.5 ml) prepared by mixing 5 ml of 30% hydrogen peroxide (Sigma-Aldrich) with 45 ml of phenol formate solution (88% formic acid and 0.5% phenol). After incubation at 0°C for 12 hours, ice-cold H2O (1 ml) was added, and the mixture was evaporated to dryness under a nitrogen stream. Subsequently, 1 ml of 6N HCl containing 0.1% phenol was added, and the sample was hydrolyzed at 110°C for 20 hours. After hydrolysis, the sample was evaporated to dryness, reconstituted with 1000 μL of H2O, and further diluted to 1 / 10. Derivatization was performed according to the AccQ-Tag method by Waters (Milford, MA, USA).

[0183] To determine the presence of tryptophan, the sample was hydrolyzed under alkaline conditions using a 4M lithium hydroxide (LiOH) solution containing ascorbic acid (95 mM). A lyophilized sample (2 mg, accurately weighed) was dissolved in 0.9 ml of freshly prepared hydrolysis solution. Vacuum was applied to the hydrolysis tube, followed by nitrogen purging of the headspace. The sample was hydrolyzed at 110°C for 16 hours. After hydrolysis, the sample was neutralized by adding 600 ml of hydrochloric acid (6 M), and the sample was derivatized as described above by adding an internal standard of methyl-tryptophan.

[0184] UPLC Analysis: Analysis was performed on a Waters (Milford, MA, USA) Acquity UPLC system equipped with a diode array detector. Chromatography was performed using a Waters (Milford, USA) Acquity Mass TRAK tm (2.1 × 100 mm, 1.7 μm) column, maintained at 43°C. The injection volume was 1 μL. Separation was performed at a flow rate of 0.4 mL / min using a gradient elution program. The signal was detected at 260 nm. Standard materials were derivatized in the same manner as the samples.

[0185] His, Ser, Arg, Gly, Asp, Glu, Thr, Ala, Pro, Cys, Lys, Tyr, Met, Vai, Ile, Leu, Phe, and Trp were quantified in the sample. In acid hydrolysis, Asn was converted to Asp and Gin to Glu. Cysteine ​​and methionine were identified as cysteic acid and methionine sulfone after oxidation. The protein content of the sample can be estimated based on the sum of the identified amino acids.

[0186] Table 13 reports the protein content of cultured avocado cells with and without Gly+Glu supply, determined according to the analytical method described above. The control refers to cultured avocado cells without Gly+Glu supply.

[0187] TIFF2026521485000014.tif33145

[0188] As shown in Table 13, the total amino acid content was observed to increase by 22% (p<0.01, T-test) when Gly+Glu was supplied compared to a control culture without Gly+Glu. In other words, avocado cells recovered from a culture supplied with Gly+Glu had 22% more protein content than avocado cells from a control culture. This increase in protein content is particularly beneficial when using the entire cultured avocado cell without extracting the compound. Figure 15 shows the analyzed individual amino acid content of cultured avocado cells with and without Gly+Glu supply. Figure 15 shows that, compared to a control culture without Gly+Glu supply, all amino acids increased or remained at the levels of the control culture in cultured avocado cells supplied with Gly+Glu.

[0189] The supply of Gly+Glu resulted in a 16% reduction in recovered biomass. However, even considering the reduction in biomass, the supply of Gly+Glu resulted in a 3% increase in total protein yield. However, when using whole cultured avocado cells, the protein content of the recovered biomass may be a more interesting feature than the total protein yield.

[0190] Example 6 - Sensory evaluation of cultured avocado cells Avocado cells were cultured in a shaking flask for 12 days as described in Examples 1 and 2, filtered through a miracloth, and washed with sterile water. The collected cultured avocado cells were stored in a freezer. A portion of the cultured avocado cells were treated, i.e., lysed, by microfluidization.

[0191] Microfluidization or dissolution was performed in a food-grade facility. In addition to the buckets and tools used for dissolution, the microfluidizer was cleaned following a careful cleaning procedure. First, the containers were washed with dish soap and tap water. Then, both the containers and the microfluidizer were washed with ethanol and then thoroughly rinsed with reverse osmosis (RO) water before starting microfluidization. Dispersions were prepared from thawed cultured avocado cells by using purified reverse osmosis (RO) water. The thawed cultured avocado cell aggregates were diluted with RO water in a 50:50 w:w ratio. The sample size was 115 g. Cell disruption (lysis) was performed by feeding the dispersion into a microfluidizer, a Microfluidics M110-EH with two Z-chambers, after pre-mixing. Pre-mixing was performed by dispersing for approximately 15 seconds using a Waring blender, or by pre-mixing the sample in a microfluidizer by passing it once through a 400 μm chamber of the microfluidizer at an operating pressure of 400 bar. In the actual cell disruption step, the sample was passed once through a microfluidizer chamber with a diameter of 400 μm at an operating pressure of 1500 bar. Output samples were collected in a clean sample container.

[0192] In addition to cultured avocado cells that were not dried, lysed, and still fresh avocado cells were evaluated for their aroma and flavor characteristics using general descriptive analysis. The mouthfeel of the cultured avocado cell samples was also investigated. Evaluations were conducted in a sensory testing laboratory by trained panelists.

[0193] A lexicon generation session was held with a small panel of four panelists, and a panel training session was held with eight panelists. The product or sample was evaluated twice (two sessions with a one-hour interval) with the participation of the eight panelists in each session. The evaluation was performed on a linear numerical scale from 0 to 10. The sample was evaluated after tasting a reference sample to match the scale.

[0194] Table 14 reports the aroma and flavor characteristics obtained from undried (fresh) cultured avocado cells and lysed undried (fresh) avocado cells.

[0195] TIFF2026521485000015.tif52164

[0196] As can be inferred from Table 14 above, fresh cultured avocado cells and lysed fresh cultured avocado cells possessed both pleasant aroma and flavor characteristics, including the flavor characteristics of avocado. Based on sensory evaluation of mouthfeel, it was observed that freeze-drying significantly improved the mouthfeel of the cells. Freeze-drying reduced undesirable sensory characteristics such as granularity (the presence of small granular particles similar to the texture of sand in the mouth), perceptibility (whether the sandy mouthfeel is easily detected), and particle size (the size of granules or particles present in the product), while the uniformity of desirable sensory characteristics (whether the sandy texture is consistent throughout the product, regardless of whether it is clustered in specific areas or uniformly dispersed) improved to a statistically significant degree compared to the corresponding fresh (undried) cells, for example, when comparing lysed fresh cells with lysed freeze-dried cells.

[0197] Various embodiments have been demonstrated. It should be understood that, in this specification, the words comprise, include, and contain are each used as open-ended expressions not intended to be exclusive.

[0198] The foregoing description provides a complete and useful description of the best mode currently intended by the inventors to carry out the invention, as non-limiting examples of specific implementations and embodiments. However, it will be apparent to those skilled in the art that the invention is not limited to the details of the embodiments shown above and can be carried out in other embodiments using equivalent means or in various combinations of embodiments without departing from the features of the invention.

[0199] Furthermore, some of the features of the embodiments disclosed in the foregoing can be advantageously used without correspondingly using other features. Therefore, the foregoing description is merely illustrative of the principles of the present invention and is not intended to limit it. Accordingly, the scope of the present invention is limited only by the appended claims.

Claims

1. Cultured avocado cells, comprising at least 15% by weight of protein based on the total dry weight of the cultured avocado cells.

2. Based on the total dry weight of the cultured avocado cells, 15-30% by weight, preferably 18-30% by weight of protein, and / or Cultured avocado cells according to claim 1, comprising 0.5 to 10% by weight, preferably 1 to 7% by weight, more preferably 1.5 to 6% by weight, and even more preferably 2 to 5% by weight of lipids.

3. The cultured avocado cells according to claim 1 or 2, wherein the cultured avocado cells contain, based on the total dry weight of the cultured avocado cells, at least 5 mg / g, preferably 5 to 25 mg / g, more preferably 7 to 20 mg / g, more preferably 10 to 18 mg / g, and even more preferably 10 to 15 mg / g of polyunsaturated fatty acids, and optionally, based on the total dry weight of the cultured avocado cells, the linoleic acid (C18:2n6) content in the cultured avocado cells is at least 5 mg / g, for example 5 to 20 mg / g, preferably at least 8 mg / g, more preferably 8 to 15 mg / g, and even more preferably 8 to 10 mg / g, and / or the alpha-linoleic acid (C18:3n3) content is 1 mg / g or more, preferably 1.50 mg / g or more, more preferably 1 to 3 mg / g, and even more preferably 1.5 to 2 mg / g.

4. The cultured avocado cells according to any one of claims 1 to 3, wherein the cultured avocado cells contain 3 to 15 mg / g, preferably 5 to 10 mg / g, saturated fatty acids and / or less than 10 mg / g, preferably less than 5 mg / g, more preferably 1 to 3 mg / g, monounsaturated fatty acids based on the total dry weight of the cultured avocado cells, and optionally, the oleic acid (C18:1) content in the cultured avocado cells is less than 5 mg / g, preferably up to 2 mg / g, based on the total dry weight of the cultured avocado cells.

5. The cultured avocado cells according to any one of claims 1 to 4, comprising 10 to 65% by weight, preferably 14 to 65% by weight, more preferably 25 to 64% by weight, and even more preferably 55 to 64% by weight of carbohydrates, based on the total dry weight of the cultured avocado cells.

6. The cultured avocado cells according to any one of claims 1 to 5, wherein the cultured avocado cells contain 0.5 to 2.0 mg / g, preferably 0.5 to 1.5 mg / g, more preferably 0.75 to 1.25 mg / g of sterols based on the total dry weight of the cultured avocado cells, and optionally, the β-sitosterol content in the cultured avocado cells is 0.5 to 1.0 mg / g based on the total dry weight of the cultured avocado cells.

7. The cultured avocado cells are substantially free of caryophyllene and / or tocopherol, and / or The cultured avocado cells according to any one of claims 1 to 6, wherein the cultured avocado cells contain (one or more) catechins and / or (one or more) procyanidins, preferably (one or more) catechins and / or (one or more) oligomeric procyanidins.

8. The cultured avocado cells according to any one of claims 1 to 7, wherein the cultured avocado cells are in the form of a cell lysate or powder, preferably a powder obtained by freeze-drying and / or spray-drying, more preferably freeze-drying, followed optionally by mechanical powdering such as homogenization, grinding and / or crushing.

9. The cultured avocado cells according to any one of claims 1 to 8, wherein the cultured avocado cells are food-grade avocado cells for human consumption.

10. A method for producing cultured avocado cells, The process of providing avocado cells, The steps include: initiating cell suspension culture by seeding the provided avocado cells into a liquid medium, and culturing the avocado cells in the cell suspension culture for a sufficient time and under conditions to result in a protein content of at least 15% by weight based on the total dry weight of the avocado cells; A method comprising the step of collecting avocado cells from the cell suspension culture to obtain cultured avocado cells.

11. The aforementioned liquid culture medium At least one cytokinin or cytokinin-like plant growth regulator (CN), preferably selected from kinetin, 6-(Y,y-dimethylallylamino)purine, zeatin, or any combination thereof, and The material comprises at least one auxin (AUX) selected from α-naphthaleneacetic acid, indoleacetic acid, or any combination thereof, preferably α-naphthaleneacetic acid. The method according to claim 10, wherein the CN concentration in the liquid culture medium is preferably 0.1 to 5.0 ppm, and the AUX concentration in the liquid culture medium is preferably 1.0 to 10 ppm.

12. The method according to claim 10 or 11, further comprising the step of carrying out the cell suspension culture in an atmospheric environment.

13. The liquid culture medium contains glycine (Gly) and glutamic acid (Glu) preferably at a concentration of 0.6 to 5.0 mM (millimoles / dm³). 3 The method according to any one of claims 10 to 12, wherein the total amount of the substance is preferably 0.8 to 3.0 mM, more preferably 1.0 to 2.0 mM.

14. The process of providing avocado cells, The process of growing callus from avocado on a solid growth medium, The method according to any one of claims 10 to 13, comprising the step of providing avocado cells from the callus.

15. The process of providing avocado cells, A step of selecting avocado cells that produce polyunsaturated fatty acids in cell suspension culture, wherein the avocado cells are preferably selected from the callus. The process includes the step of providing the selected avocado cells, The culture in the aforementioned cell suspension culture, The method according to any one of claims 10 to 14, comprising the step of culturing the cultured avocado cells for a sufficient time and under conditions to result in a polyunsaturated fatty acid content of at least 5 mg / g based on the total dry weight of the cultured avocado cells.

16. The method according to any one of claims 10 to 15, wherein the culture time and conditions of the cell suspension culture are selected to result in a lipid content of 0.5 to 10% by weight, preferably 1 to 7% by weight, more preferably 1.5 to 6% by weight, and even more preferably 2 to 5% by weight, based on the total dry weight of the cultured avocado cells, and / or substantially free of caryophyllene and / or tocopherol.

17. The method according to any one of claims 10 to 16, wherein the culture time in the cell suspension culture is in the range of 4 to 20 days, preferably 7 to 15 days, more preferably 7 to 12 days or 7 to 11 days.

18. The method according to any one of claims 10 to 17, comprising the step of using the cultured avocado cells in or as food for human consumption.

19. The method according to any one of claims 10 to 18, wherein the method comprises a step of pulverizing the cultured avocado cells, the step preferably comprising drying the cultured avocado cells preferably by freeze-drying and / or spray-drying, more preferably by freeze-drying, and subsequently optionally by mechanical pulverization such as homogenization, pulverization and / or crushing.

20. The method according to any one of claims 10 to 19, wherein the callus and the cultured avocado cells consist substantially of undifferentiated cells.

21. The method according to any one of claims 10 to 20, wherein the callus grows from avocado fruit material or avocado plant section, preferably from a section or multiple sections from an avocado seed, and the seed is optionally subjected to germination before cutting.

22. A food containing cultured avocado cells according to any one of claims 1 to 9.

23. The food according to claim 22, comprising one or more food-grade additives, preferably one or more of (one or more) thickeners, (one or more) antioxidants, (one or more) colorants, and / or (one or more) flavorings.

24. The food according to claim 22 or 23, comprising (one or more) 1 to 5% by weight of a thickening agent and / or (one or more) 1 to 4% by weight of an antioxidant, based on the total weight of the food.

25. The food according to any one of claims 22 to 24, wherein the food is a nutritional supplement, spread, dip, guacamole, flakes, dumplings, squeezed biomass for sushi, muesli, granola, or smoothie.

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