阅读说明：本技术 用克鲁维毕赤酵母生产不含酒精的发酵蔬菜汁 (Production of alcohol-free fermented vegetable juice by using pichia kluyveri ) 是由 S·萨伦 J·H·斯维格斯 于 2019-07-23 设计创作，主要内容包括：本发明涉及制备蔬菜汁的方法,包括用克鲁维毕赤酵母发酵蔬菜基质。根据该方法获得的发酵蔬菜汁风味特征更好,特别是土腥风味减少。本发明还提供了可用于发酵蔬菜基质的克鲁维毕赤酵母菌株,可以进一步加工该蔬菜基质以获得饮料或其他消费品。(The present invention relates to a method for preparing vegetable juice comprising fermenting a vegetable substrate with pichia kluyveri. The fermented vegetable juice obtained by the method has better flavor characteristics, especially reduced earthy flavor. The invention also provides pichia kluyveri strains that can be used to ferment vegetable substrates that can be further processed to obtain beverages or other consumable products.)

1. The method for reducing the fishy smell and the peculiar smell of the vegetable juice comprises the following steps:

a) providing a substrate comprising a root vegetable,

b) adding at least one Pichia kluyveri (Pichia kluyveri) strain to the substrate,

c) fermenting a substrate comprising the Pichia strain,

d) obtaining the fermented vegetable juice.

2. The method of claim 1, wherein the root vegetable is selected from the group consisting of: carrot, beetroot, parsnip, swedish cabbage, turmeric, ginger, potato, sweet potato, turnip cabbage, cassava, cabbage, onion, garlic, celery root, horseradish, white radish, yam bean, radish, jerusalem artichoke, and yam.

3. The method of any one of the preceding claims, wherein the Pichia kluyveri is selected from the group consisting of:

a) pichia kluyveri, deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), given deposit number DSM28484,

b) pichia kluyveri, deposited at 24.8.2006 at the national institute for measurements and research, Okland university, Okland's institute for Life sciences, Okland, 1142, New Zealand at No. 541-65, southern Melbourne street, 3205, Australia, under accession number V06/02271,

c) pichia kluyveri, deposited at the national institute for measurements and research at 3205 south melbourne clarke street No. 541-65, national institute for bioscience, Okland university, Okland 1142, New Zealand, Okland 24.2006, assigned accession number V06/02272, and

d) a), b) or c).

4. The process according to any one of the preceding claims, wherein the weight ratio of isoamyl acetate to ethyl acetate is at least 0.1, preferably at least 0.2, and/or wherein the weight ratio of isoamyl acetate to ethanol is at least 0.0002, preferably at least 0.0008.

5. The method of any one of the preceding claims, wherein step b) further comprises adding a strain of lactic acid bacteria to the matrix.

6. The process according to any of the preceding claims, wherein the fermentation in step c) is carried out at a temperature of 4-35 ℃, more preferably 18-22 ℃.

7. The method of any of the preceding claims wherein the beta-phenylethyl alcohol in the fermented vegetable juice is at least 0.4 ppm.

8. The method of any preceding claim wherein the alpha-terpineol in the fermented vegetable juice is at least 50 ppb.

9. The process of any preceding claim, wherein the c-whiskey lactone or vanillin in the fermented vegetable juice is at least 4 ppb.

10. Fermented vegetable juice obtained by the method according to any of the preceding claims, wherein the juice comprises Pichia kluyveri.

11. Fermented vegetable juice comprises root vegetables and Pichia kluyveri.

12. The fermented vegetable juice of claim 11 wherein the pichia kluyveri is selected from the group consisting of:

a) pichia kluyveri, deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), given deposit number DSM28484,

b) pichia kluyveri, deposited at 24.8.2006 at the national institute for measurements and research, Okland university, Okland's institute for Life sciences, Okland, 1142, New Zealand at No. 541-65, southern Melbourne street, 3205, Australia, under accession number V06/02271,

c) pichia kluyveri, deposited at the national institute for measurements and research, No. 541-65, 3205 south melbourne street, Okland, 24 D.C. 2006, Okland university, Okland 1142, Victoria, Australia, under accession number V06/02272, and

d) a), b) or c).

13. Application of Pichia kluyveri in fermenting root vegetable is disclosed.

14. The use of claim 13, wherein the pichia kluyveri is selected from the group consisting of:

a) pichia kluyveri, deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), given deposit number DSM28484,

b) pichia kluyveri, deposited at 24.8.2006 at the national institute for measurements and research, Okland university, Okland's institute for Life sciences, Okland, 1142, New Zealand at No. 541-65, southern Melbourne street, 3205, Australia, under accession number V06/02271,

c) pichia kluyveri, deposited at 24.8.2006 by the Okland university Life sciences college of Okland, Okland 1142, New Zealand at the national institute of measurements at 3205 south Melbourne street 541-65, Victoria, Australia, under accession number V06/02272, and

d) a), b) or c).

15. Pichia kluyveri, which was deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), and was assigned the deposit number DSM28484, and mutants thereof.

Technical Field

The present invention relates to low-alcohol or alcohol-free beverage products of plant origin, in particular to vegetable juice products. The invention also relates to the use of microorganisms for preparing such beverages.

Background

Vegetables are an important dietary source of important nutrients required by the human body. Vegetable juice based diets are becoming increasingly popular. Vegetable juice is considered a healthy alternative to fruit juice because it contains less sugar. However, vegetable juices have the problem that the flavour profile is not always appealing. The taste of fresh vegetable juice may be earthy (e.g. beetroot juice, carrot juice and cabbage juice) or sulphur (e.g. leek juice and onion juice), which is the main reason why vegetable juices are not as palatable as fruit juices. It has been a challenge to produce vegetable juices that are acceptable to a wide range of consumers, involving balancing flavor, aroma, appearance, and satisfying mouthfeel.

In addition, when the vegetable juice is heat-treated by pasteurization for long-term preservation, more off-flavors are generated or enhanced in the process. Certain off-flavors are described as cooked vegetables that are not appealing to consumers. To address this problem, the juice industry often mixes vegetable juice into the juice.

Fermentation processes have been used to improve the taste of beverages. Corona et al (Corona, Onoforo, et al, "chromatography of kefir-like catalysts produced from transformed sources," LWT-Food Science and Technology 66(2016): 572-. The Lactobacillus consists of Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus kefir and Lactobacillus fermentum. The fermented vegetable and fruit juices were analyzed in a sensory panel of 15 untrained panelists. The only juice with the desired flavor profile is the carrot kefir-like beverage. However, the carrot kefir-like beverage has an alcohol content of more than 3%, which is undesirable for a healthy beverage.

CN108244425 discloses a functional beverage prepared by fermenting beetroot (Beta vulgaris) juice with lactic acid bacteria and blending with tomato juice. Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus rhamnosus (Lactobacillus rhamnosus) and Lactobacillus fermentum are used for the fermentation of beetroot juice. Other ingredients including lemon extract, peppermint extract, anise extract, honey and isomalto-oligosaccharide were added to impart aroma and refreshing taste.

WO2012066176 describes the production of alcoholic fermented orange juice by using the Pichia kluyveri (Pichia kluyveri) strain deposited as CECT 13055. The strain is capable of fermenting reducing sugars present in orange juice, so that the final product contains non-reducing sugars, such as sucrose, thereby providing a pleasant tasting product. Furthermore, the alcohol content of the final product is typically about 2.5%.

WO2011134952 discloses the inoculation of pichia kluyveri into grape juice. The yeast added in frozen form is thawed to liquid form prior to addition to the culture medium. It has been found that frozen yeast can be used for direct inoculation without significant loss of cell count and subsequent activity. This document does not describe any improvement in the taste of the beverage.

US20170258113 discloses a method for preparing a bayberry fermented juice using pichia kluyveri XT 110. As disclosed, pichia kluyveri XT110 is capable of fermenting at pH 2.5-3.0, which is important for keeping anthocyanins stable and therefore does not cause severe discoloration of the final product. The prepared waxberry fermented juice has bright color, high anthocyanin content, strong fruit flavor, balanced flavor and low alcohol content (less than 0.5%).

WO2015117978 discloses the preparation of cider from apple juice and/or pear juice using two Pichia kluyveri strains PK-KR1 and PK-KR 2. The product was found to contain increased amounts of hexyl acetate and other desirable flavor compounds, particularly hexyl acetate and isobutyl acetate. These two Pichia kluyveri strains PK-KR1 and PK-KR2 were originally disclosed in WO2009110807, which describes their use for increasing thiol levels (3MH and 3MHA) in wine fermentation processes. 3MH and 3MHA have unique grapefruit and passion fruit-like aromas and flavors. PK-KR1 and PK-KR2 are also used in brewing beer with different hop varieties (WO2013030398) and low-alcohol or alcohol-free beer (WO 2014135673).

However, there is no solution to enhance the flavor of vegetable juices. New methods for preparing vegetable juices having desirable flavor characteristics, such as reduced undesirable off-flavors, are needed.

Disclosure of Invention

The present invention provides a novel process for preparing flavor-enhanced vegetable juice. It has been found that pichia kluyveri can be advantageously used for fermenting vegetable juices to obtain products with improved flavor characteristics and thus palatability.

As previously mentioned, the flavor characteristics of vegetable juices are not always appealing as opposed to fruit juices. Thus, the present invention provides different methods of improving the flavor of vegetable juices without the need for mechanical techniques or blending the juices.

In particular, it has been found that pichia kluyveri can be used to produce increased levels of flavor compounds, including isoamyl acetate, isobutyl acetate, β -phenylethyl alcohol, α -terpineol, c-whiskey lactone, and vanillin.

Sensory evaluation clearly showed that in vegetable juice fermented with pichia kluyveri much less fishy off-flavour was perceived. Fermented juices also have more fruity and pungent flavors than unfermented juices, and are generally preferred.

In a first aspect, the present invention provides the use of pichia kluyveri for the preparation of a beverage comprising root vegetable material. It has been found that pichia kluyveri can be advantageously used for treating vegetable juices, in particular vegetable juices having an undesirable earthy flavour. Fishy flavors are common for vegetable juices prepared from plant materials that grow in or in contact with soil. Therefore, it is desirable to enhance the flavor of vegetable juices by reducing earthy taste. Thus, the present invention provides another strategy to improve flavor profile that is different from the methods currently used in the industry that involve the use of mechanical techniques or mixing in juices.

Preferred pichia kluyveri strains include pichia kluyveri strain 1, pichia kluyveri strain 2 and pichia kluyveri strain 3 and mutants thereof.

In another aspect, the invention provides the use of pichia kluyveri to reduce the earthy flavor of plant materials. The use relates to fermenting plant material, in particular root vegetables.

In another aspect, the present invention provides a method of treating a vegetable juice to reduce its earthy flavour and vegetable juices obtained thereby. In one embodiment, the method comprises the steps of: (a) providing a substrate comprising root vegetable material, (b) adding at least one pichia kluyveri strain to the substrate, (c) fermenting the substrate, and (d) obtaining a fermented vegetable juice. It should be understood that the substrate may include more than one type of root vegetable.

In other aspects, the invention also provides vegetable juices obtained by the methods described herein, as well as vegetable juices comprising one or more pichia kluyveri strains, such as the pichia kluyveri strains described herein.

The invention also provides a new strain of Pichia kluyveri, which was deposited at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ) 7B, D-38124 on 3.5.3.2014, and which was assigned the deposit number DSM 28484.

Definition of

As used herein, the term "vegetable" refers to the edible portion of a plant for human consumption. The edible parts of the plant may be roots, such as rutabaga, beet, carrot and sweet potato; tubers or storage stems, such as potatoes and taros; stems, such as those of asparagus and cabbage (kohlrabi); sprouts, such as Brussels sprouts (Brussels sprouts); bulbs, such as onion and garlic; petioles (pelioles) or leafstalks (leaftalk), such as celery and rhubarb; leaves, such as cabbage, lettuce, parsley and spinach; immature flowers, such as broccoli, broccoli and artichoke.

As used herein, the term "root vegetable" refers to the edible portion of a plant vegetable that comes into contact with the soil. Root vegetables are usually storage organs that are enlarged to store energy in the form of carbohydrates. It should be noted that "root vegetables" as used herein is not limited to botanically defined roots, but also includes true roots (e.g., main roots and tuberous roots) and stems (e.g., bulbs, corms, rhizomes, and tubers).

Generally, "off-flavor" refers to the presence of an objectionable taste and/or odor in a food product. "earthy" is a flavor similar to soil. Since this flavor is undesirable in food products, it is also referred to herein as "earthy off-flavor". Soil odor is largely attributed to the presence of Geosmin (Gerber, n.n., and h.a.lechevalier. "Geosmin, an earth-compacting substance isolated from microorganisms." Applied microbiology 13.6(1965): 935-938). Soil odor pixels in soil have been characterized (Buttery, Ron g., and John a. garibaldi. "Geosmin and methylisoborneol in garden soil." Journal of Agricultural and Food Chemistry24.6(1976): 1246-.

However, since a specific apparatus is required, it is difficult to measure the geosmin. However, the earthy flavour caused by earthy flavourants can be reliably assessed using sensory analysis known in the art. Thus, as used herein, "reducing the earthy taste" refers to reducing the degree of perception of earthy taste. This can be done by comparing the native flavor before and after treatment with pichia kluyveri. This can be assessed by sensory evaluation methods known in the art, such as those described by Meilgaard et al (Meilgaard, Morten C., B.Thomas Carr, and Gail Vance Civille. Sensory evaluation techniques. CRC press,1999) or described herein.

As described in the examples, the earthy flavour can be measured by sensory analysis.

The term "substrate" refers to a material that is fermentable by pichia kluyveri.

The term "alcohol free" refers herein to an alcohol content of less than 0.5% v/v.

The term "enhancing" the "flavor" of a product refers to improving the flavor profile of the product so that it becomes more palatable. This can be determined, for example, by sensory evaluation known to those skilled in the art.

The term "fermentation" generally refers to any activity or process involving the enzymatic breakdown (digestion) of organic material by microorganisms. The term "fermentation" encompasses anaerobic and aerobic processes, as well as processes involving a combination or succession of one or more anaerobic and/or aerobic stages.

The term "mutant" is to be understood as a strain derived from a strain of the invention by means of, for example, genetic engineering, radiation and/or chemical treatment. Preferably, the mutant is a functionally equivalent mutant, e.g. a mutant having substantially the same or improved properties as the mother strain. In the context of the present invention, the mutant of the invention is preferably a mutant having the same or improved properties in terms of reduction of the earthy taste. Such mutants are part of the present invention. The mutant may be a strain obtained by subjecting the strain of the present invention to any conventionally used mutagenesis treatment including treatment with a chemical mutagen such as Ethane Methane Sulfonate (EMS) or N-methyl-N' -nitro-N-Nitroguanidine (NTG), ultraviolet treatment, or a spontaneously occurring mutant. The mutant may have undergone several mutagenic treatments (a single treatment should be considered a mutagenic step followed by a screening/selection step), but it is presently preferred that no more than 1000, no more than 100, no more than 20, no more than 10 or no more than 5 treatments are performed. In preferred mutants of the invention, less than 5% or less than 1%, or even less than 0.1% of the nucleotides in the bacterial genome have been altered (e.g. by substitution, insertion, deletion or a combination thereof) compared to the mother strain.

Drawings

FIG. 1 shows a schematic view of a

Sensory analysis of beetroot juice prepared by fermentation with Pichia kluyveri strain 1 and Pichia kluyveri strain 2.

Detailed Description

The invention belongs to the technical field of alcohol-free beverages. It will be appreciated that the ethanol concentration of such beverages is less than 0.5% (v/v).

The non-alcohol and low alcohol sectors are rapidly growing driven by health-conscious consumers seeking to limit alcohol consumption. In particular, vegetable juices are becoming increasingly popular among health-oriented consumers. However, the flavor characteristics of vegetable juices are not always appealing. Thus, there is a continuing need to provide products with improved flavor profiles for the market.

In a first aspect, the present invention provides a process for obtaining fermented vegetable juice, the process comprising: adding at least one pichia kluyveri strain to a substrate comprising root vegetables, and fermenting the substrate to obtain a fermented vegetable juice. The present invention is based on the following findings: pichia kluyveri has beneficial properties that can be used to treat vegetable juices, especially to reduce earthy off-flavors and to enhance fruit and floral flavors.

In a preferred embodiment, the method comprises:

a) providing a substrate comprising a root vegetable,

b) adding at least one Pichia kluyveri strain to the substrate, and

c) fermenting a substrate comprising the Pichia kluyveri strain.

In another preferred embodiment, the method comprises:

a) providing a substrate comprising a root vegetable,

b) the pH value of the matrix is adjusted,

c) adding at least one Pichia kluyveri strain to the substrate, and

d) fermenting a substrate comprising the Pichia kluyveri strain.

In a second aspect, the present invention also provides a fermented vegetable juice obtained by the method described herein.

The fermented vegetable juice is free of alcohol. For example, the alcohol content (v/v) of the fermented vegetable juice is less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.2%, such as less than 0.1%, such as less than 0.09%, such as less than 0.08%.

Pichia kluyveri is expected to be particularly useful for processing all vegetables with an earthy flavor. This includes, in particular, root vegetables.

The earthy flavor is the characteristic feature of many vegetable juices and is caused by aromatic compounds earthy flavor. Geosmin (trans-1, 10-dimethyl-trans-9-decalin) is a compound with a very pronounced earthy, musty, beetroot, and even turnip (turnip) flavour, and has a very low sensory threshold as low as 10 ppm. It is a metabolite of various actinomycetes and fungi. It has been found to be responsible for the fishy smell of various foods, including fish and wine. It has been reported that certain bacteria such as Bacillus subtilis and Pseudomonas species can biodegrade osmylin. However, fermentation with pichia kluyveri has never been reported to reduce the perception of off-flavors from osmyl.

To prepare the vegetable juice according to the invention, a matrix comprising root vegetables is provided.

Root vegetables may be derived from one or more types of vegetables. For example, the matrix may comprise material from one particular root vegetable or a different type of root vegetable. In addition, the matrix may additionally contain other plant materials. For example, the matrix may comprise a mixture of root vegetables and fruits. In another embodiment, the matrix consists essentially of root vegetables.

The substrate according to the invention comprises root vegetables. For example, a root vegetable is any one of the known types of root vegetables. Suitable root vegetables include, but are not limited to, true roots, such as tuberous and main roots, and non-roots, such as tubers, rhizomes, corms, and bulbs.

The true root vegetables may be the main root and/or the tuberous root. Examples of the primary root include, but are not limited to: white potatoes (yellow potato (aralia xanthorrhiza)), white potatoes (bush carrot) (Abelmoschus moschatus)), sugar and fodder beets (Beta vulgaris), turnip cabbage and turnip (Brassica species) (Brassica spp.), black fennel (black cumin) (platen moss (Bunium persicum)), burdock (burdock) (Arctium), carrot (Daucus carota subsp. sativus), white root celery (pariphyllum) (Apium graveolens (Apium gracile)), white radish (daikon) (radish (Raphanus sativum)), dandelion (paradelion) (dandelion species (Taraxacum. crispum), white potato (sweet potato) (marjoram)), white potato (sweet potato (potato) (carassima purpurea), white potato (sweet potato (potato) (caraway)), white potato (sweet potato (potato), potato (potato) (milfoil), and potato (sweet potato (potato) (milfoil), potato (milfoil), radish (potato (milfoil), radish (potato (sweet potato (milfoil), and potato (sweet potato (milfoil) species (milfoil), and potato (sweet potato (milfoil) species) of Brassica), and radish (sweet potato) species) of Brassica), radish (sweet potato) of the genus) variety) of the genus) variety, parsley root (parsley root) (parsley seed (Petroselinum spp.)), radish (radish) (radish (Raphanus sativus)), black skin salsify (black salsify) (Scorzonera hispanica), parsley (skirret) (parsley sisarum), garlic leaf salsify (salsify) (gorgon spp.)) and clump of potatoes (bush potatoo) (Vigna punctata (Vigna lancelloata)).

Examples of tuberous roots include, but are not limited to, yellow lily yam (yellow lily yam) (amorphophalus galbra), groundnut (pigout) or peanut (earthmut) (Conopodium majus)), yam (nageimo), Dioscorea opposita (Chinese yam), korean yam (Dioscorea opposita)), ginger (native girald ginger) (Hornstedtia scottiana), sweet potato (Ipomoea batatas)), desert yam (Ipomoea costata), cassava (cassava) or cassava (yuca) or manioc) (cassava (Manihot esculenta)), mauka or chargo mirabilis extana, edible (breava), Psoralea or grass (ariella) (saussurea), and edible (fruit) (saussurea involucrate (fruit)).

An example of a root vegetable comprising a rhizome is arrowroot florida (Zamia pumita).

Non-roots include tubers, rhizomes, corms, and/or bulbs. Examples of bulbs include, but are not limited to, konjac (Amorphophalus konjac), taro (Colocasia esculenta), chufa (Eleocharis dulcis), Eleocharis aethiopica (enset) (like plantain (Enterate spp)), nymphae (water lily) (Nelumbo nucifera), arrowhead (arowhoad) or arrowhead (wapato) (species of the genus Sagittaria spp.), arundina elatus (yatia) or russiana (maranga) (species of the genus Xanthocerana (Xanthomonas spp.)).

Examples of rhizomes include, but are not limited to, turmeric (Curcuma longa), ginseng (Panax ginseng)), lilium crispum (rengarengga), and lilium vanicum (vanilla lily) (species of lilium genus of longuey (artropium spp.)), Canna (Canna) (species of Canna spp.)), plantain (Cordyline utcosa), arrowroot (arrowroot) (arrowroot (candinaceae)), lotus root (lotus root) (Nelumbo nucifera), caraway (or reed (bulbus (typhaline utcosa)), ginger (galangal) (Zingiber officinale)).

Examples of tubers include, but are not limited to, porcine potato (hog potato) or groundnut (groundnut) (Apios americana), Cyperus esculentus (tigernut), or Cyperus rotundus (chufa); yam, ube (Dioscorea species (diosorea spp.)), Jerusalem artichoke (Jerusalem artichoke) or Jerusalem artichoke (sunchoke) (Jerusalem artichoke (Helianthus tuberosus)), daylily (Hemerocallis spp.)), rhododendron mollissima (earhnu pea) (lathyrius rhododendron (lausuryrus tuberosus)), New Zealand yam (New Zealand yam) and pyrola rotundifolia (oca) (potato Oxalis tuberosa), kembii, dazo (pignathus sativus and south africana), potato (Solanum tuberosum), Chinese honeylocust (chinarum tuberosum) or sweet potato (mannose) (mannan (sunshinus), tuber (lotus), or lotus seedpod (lotus), or lotus seed (lotus seed) or tuber (anu)).

Examples of bulbs include, but are not limited to, garlic, onion, shallot, leek, etc. (Allium spp.),. Ipomoea batatas (Lilium furfur), sympodial onion (Bush onion) (Cyperus bulbosus), Sagitaria japonica (katakuri) (Erythronium spp.),. Limoni (fennel) (Foeniculum vulgare)) and Lily (lilies) (Lily spp.)).

The nutritional composition of root vegetables, such as the composition of carbohydrates, may vary. For example, tubers contain more starch than real root vegetables, which may contain more monosaccharides. Both true roots and non-roots are sources of other vitamins, minerals and other nutrients, such as fiber, potassium, vitamin a, calcium, vitamin B6, vitamin C, biotin, vitamin B1, vitamin B2, vitamin B3, iron, copper and magnesium.

Suitable root vegetables for preparing the vegetable juice may be monocotyledonous, dicotyledonous, or combinations thereof. Examples of monocots ("monocots") ") include yams, such as White Yam (White Yam), Yellow Yam (Yellow Yam), Kokoro, western Yam (Japanese Mountain Yam), Purple Yam (Purple Yam), Yam beans (Jicama), Yellow Yam, or combinations thereof.

Examples of dicotyledonous plants include sweet potato, Batas, Brazilian sweet potato (Brazilian sweet potato), Dinges sweet potato (Dingess sweet potato), Japanese sweet potato (Japanese sweet potato), Okinawan sweet potato (Okinawan sweet potato), Mokuau sweet potato (Mokuau sweet potato), Kangton sweet potato (Covington sweet potato), Beauregard sweet potato (Beauregard sweet potato).

The substrate described herein may comprise, for example, one or more types of root vegetables. For example, the substrate may comprise carrot and beetroot. In some embodiments, the matrix comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different types of root vegetables.

Preferred examples of root vegetables include, but are not limited to, carrot, beetroot, parsnip, swedish (swede), turmeric, ginger, potato, sweet potato, turnip cabbage, cassava, cabbage, onion, garlic, celery root, horseradish, white radish, yam bean, radish, jerusalem artichoke, and yam.

The process of preparing the matrix can be summarized in the following manner: the vegetables are washed, optionally ground to a slurry and extracted (preferably by pressing) to a slurry-free vegetable juice. Depending on the type of vegetable, the vegetable juice may be pasteurized or sterilized, if desired. Other ingredients may also be present in the matrix.

In one embodiment, the vegetable juice may be acidified. This can be done, for example, by adding acidulants such as citric acid, lactic acid, lemon juice, and the like. Alternatively, this can be done by fermentation with lactic acid bacteria. Acidification may be used to stabilize the juice to reduce pathogen growth in the final product.

Thus, in another embodiment, the present invention provides a process comprising the steps of:

a) providing a substrate comprising a root vegetable,

b) the pH value of the matrix is adjusted,

c) adding at least one Pichia kluyveri strain to the substrate, and

d) fermenting a substrate comprising the Pichia kluyveri strain.

The pH is preferably adjusted to 2.5-6.5, for example 2.5-6 or 3-5.5. Pichia kluyveri is capable of fermenting juice in these pH ranges. The pH of the matrix can be adjusted according to the product desired by the consumer.

The method according to the invention comprises the step of adding pichia kluyveri to the substrate. This may include the addition of one or more pichia kluyveri strains, for example 1, 2, 3, 4, 5 or more pichia kluyveri strains. As used herein, a pichia kluyveri "strain" refers to a genetic variant of pichia kluyveri, which can be readily determined by one skilled in the art based on its genetic composition.

Pichia kluyveri is added to the substrate in an amount sufficient to initiate and maintain fermentation. The skilled person should be able to readily determine the appropriate inoculation concentration for pichia kluyveri, according to the description and the examples.

In a preferred embodiment, at least 1x10⁴CFU/ml, e.g. at least 5x10⁴CFU/ml, e.g. at least 1X10⁵CFU/ml, e.g. at least 5x10⁵CFU/ml, e.g. at least 1X10⁶CFU/ml, e.g. at least 5x10⁶Inoculation gram of CFU/ml concentrationPichia luviensis.

In some embodiments, a blend of more than one strain may be used, for example 2, 3, 4, 5 or more pichia kluyveri strains.

Furthermore, the pichia kluyveri yeast added can be in frozen, liquid or dried form, including for example lyophilized and spray/fluid bed dried forms or frozen or lyophilized concentrates.

The method according to the invention comprises the step of fermenting a substrate comprising the added pichia kluyveri. The fermentation of the substrate is preferably carried out by controlled fermentation under sterile conditions. During fermentation, the skilled person will be able to choose several other options known to him, such as but not limited to adding pectinase during milling/pressing, adjusting pH, adding sulphur dioxide as an antimicrobial agent, clarification and filtration, in order to obtain the desired end product.

Preferably, the fermentation of the substrate with pichia kluyveri is carried out for at least 12 hours, such as 18 hours, such as 24 hours, such as 36 hours, such as 48 hours. Preferably, the fermentation does not exceed 48 hours, as the alcohol content may increase to levels that are undesirable for the intended end product. The skilled person will be able to easily select a suitable fermentation time depending on the desired properties of the product.

In some embodiments, the fermentation is not longer than 36 hours. In other embodiments, the fermentation is conducted for 12-48 hours.

Preferably, the fermentation conditions using pichia kluyveri are semi-anaerobic. Such fermentation may start aerobically and proceed anaerobically after all oxygen has been consumed.

In some embodiments of the invention, pichia kluyveri is added to the substrate as a starter culture. The term "starter culture" refers to a composition comprising living microorganisms capable of initiating or effecting fermentation of organic material, optionally after culturing in a separate starter culture medium to obtain a high density culture. Thus, in embodiments, the starter culture of the present invention may be a high density culture obtained by propagating the starter culture in a suitable medium.

In addition to microorganisms, the starter cultures according to the invention may also contain buffers and growth-stimulating nutrients or preservatives or other carriers.

In a preferred embodiment, the first starter culture contains at least 10²Individual Colony Forming Units (CFU)/g Pichia kluyveri, e.g. at least 10³CFU/g, e.g. at least 10⁴CFU/g, e.g. at least 10⁵CFU/g, e.g. at least 10⁶CFU/g, e.g. at least 10⁷CFU/g, e.g. at least 10⁸CFU/g, e.g. at least 10⁹CFU/g, e.g. at least 10¹⁰CFU/g, e.g. at least 10¹¹CFU/g, e.g. at least 10¹²CFU/g, e.g. at least 10¹³CFU/g。

The fermentation may be carried out at a temperature of 4-35 ℃. In a preferred embodiment, the fermentation is carried out at a temperature of 18-22 deg.C, such as 19-21 deg.C, such as 20 deg.C.

The fermentation may be terminated by any suitable method known in the art, including cooling to preferably below 4 ℃ and/or pasteurization. Then obtaining fermented vegetable juice. Preferably, the fermentation broth is separated from the solids using any suitable method known in the art, for example, by filtration or centrifugation.

Depending on the product, other ingredients such as thickeners, sugars, sweeteners, dietary fibers, syrups, buffers, pH adjusters, salts, essential minerals, pectins, proteins, flavors, colorants, vitamins, preservatives, antioxidants, and the like may also be included in the fermented juice.

It is also envisioned that the fermentation broth may be further processed, for example, by concentration, dilution, or mixing with other products.

The fermented juice is preferably bottled and optionally pasteurized. In other embodiments, the fermented juice is not pasteurized.

Acetate is an important flavor compound in beverages. For example, the best known acetates are isoamyl acetate (isoamyl acetate) and ethyl acetate. Ethyl acetate is a fruity liquid with a brandy taste, an ester common in fruit. The inventors have found that after fermentation, the level of ethyl acetate in vegetable juice with pichia kluyveri is increased. In some embodiments, the level of ethyl acetate is at least 10ppm, such as at least 12ppm, at least 13ppm, at least 14ppm, at least 15ppm, at least 16ppm, at least 17ppm, at least 18ppm, at least 19ppm, at least 20ppm, at least 21ppm, at least 22ppm, at least 23ppm, at least 24ppm, at least 25ppm, at least 26ppm, at least 27ppm, at least 28ppm, at least 29ppm, or at least 30 ppm.

Isoamyl acetate, also known as isoamyl acetate (isopentyl acetate), is an organic compound and is an ester formed from isoamyl alcohol and acetic acid. Isoamyl acetate is naturally present in banana plants. It has a strong odor, which is also described as a banana and pear-like odor. However, it produces a noticeable acetone-like off-taste in large amounts, which is unpleasant to perceive.

The inventors have found that after fermentation, the level of isoamyl acetate in vegetable juice with pichia kluyveri is increased. In some preferred embodiments, the isoamyl acetate in the fermented vegetable juice prepared according to the present invention is at least 2ppm, such as at least 2.1ppm, at least 2.2ppm, at least 2.3ppm, at least 2.4ppm, at least 2.5ppm, at least 2.6ppm, at least 2.7ppm, at least 2.8ppm, at least 2.9ppm, at least 3.0ppm, at least 3.1ppm, at least 3.2ppm, at least 3.3ppm, at least 3.4ppm, at least 3.5ppm, at least 3.6ppm, at least 3.7ppm, at least 3.8ppm, at least 3.9ppm, or at least 4.0 ppm.

The weight ratio of isoamyl acetate to ethyl acetate is a good indicator of fruity taste. Like ethanol, ethyl acetate is an ester of ethanol and acetic acid that acts as a solvent. It helps to release the flavor of isoamyl acetate.

The inventors have found that this weight ratio is increased in vegetable juice fermented with pichia kluyveri after fermentation. In some preferred embodiments, the weight ratio of isoamyl acetate to ethyl acetate in the fermented juice prepared according to the present invention is at least 0.1, such as at least 0.11, at least 0.12, at least 0.13, at least 0.14, at least 0.15, at least 0.16, at least 0.17, at least 0.18, at least 0.19 or at least 0.2. Preferably, the weight ratio of isoamyl acetate to ethyl acetate in the fermented juice is at least 0.2, such as at least 0.21, at least 0.22, at least 0.23, at least 0.24, at least 0.25, at least 0.26, at least 0.27, at least 0.28, at least 0.29 or at least 0.30.

The inventors have found that such a weight ratio of isoamyl acetate to ethanol in vegetable juice fermented with pichia kluyveri is increased after fermentation. In some preferred embodiments, the weight ratio of isoamyl acetate to ethanol in the fermented juice prepared according to the present invention is at least 0.0002, such as at least 0.0003, at least 0.0004, at least 0.0005, at least 0.0006, at least 0.0007, at least 0.0008, at least 0.0009, at least 0.001, at least 0.0011, at least 0.0012 or greater.

It has been found that vegetable juice fermented with pichia kluyveri contains more flavor compounds, including beta-phenylethyl alcohol and alpha-terpineol. Beta-phenylethyl alcohol has a rose-like flavour and can act synergistically with other flavour compounds (such as esters) to enhance the overall fruity impression. The inventors have found that after fermentation, beta-phenylethyl alcohol in the vegetable juice fermented with pichia kluyveri is increased. In some preferred embodiments, the beta-phenylethyl alcohol in the fermented vegetable juice is at least 0.2ppm, such as at least 0.3ppm, such as at least 0.4ppm, such as at least 0.5ppm, such as at least 0.6ppm, such as at least 0.7 ppm.

Alpha-terpineol has a pleasant odor similar to clove. It is a terpene alcohol found in natural oils such as pine oil. It is also one of the most commercially important monoterpene alcohols in the perfume industry. The inventors have found that after fermentation, alpha-terpineol is increased in vegetable juice fermented with pichia kluyveri. In some preferred embodiments, the alpha-terpineol in the fermented vegetable juice is at least 5ppb, such as at least 10ppb, such as at least 20ppb, such as at least 30ppb, such as at least 40ppb, such as at least 50ppb, such as at least 60ppb, or such as at least 70 ppb.

c-whiskey lactone (cis-whiskey lactone), also known as (3S,4S) -3-methyl-4-octalactone, is an important component in the aroma of whiskey and other alcoholic beverages aged in oak barrels. It is sometimes added to wine as a flavoring. The inventors found that c-whiskey lactone is increased in vegetable juice fermented with pichia kluyveri after fermentation.

In some preferred embodiments, the c-whisky lactone in the fermented vegetable juice is at least 1ppb, such as at least 2ppb, for example at least 3ppb, such as at least 4ppb, for example at least 5ppb, such as at least 6ppb, for example at least 7 ppb.

Vanillin is a phenolic aldehyde, the main flavor and aroma compound in vanilla. Vanillin is more commonly used as an aromatic agent than a natural vanilla extract in foods, beverages and medicines because of its sweet taste and creamy vanilla taste. The inventors have found that after fermentation, vanillin is increased in vegetable juice fermented with pichia kluyveri. In some preferred embodiments, vanillin in the fermented vegetable juice is at least 2ppb, such as at least 3ppb, for example at least 4ppb, such as at least 5ppb, for example at least 6ppb, for example at least 7 ppb.

In another aspect, the present invention provides fermented vegetable juices having improved flavor characteristics. The vegetable juice prepared according to the present invention comprises root vegetables and pichia kluyveri, which may be, for example, pichia kluyveri strain 1, pichia kluyveri strain 2, pichia kluyveri strain 3, mutants thereof, or any combination thereof.

Pichia kluyveri strain 1 was deposited at 5.3.2014 at the German Collection of Microorganisms and cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ) of Huffman street 7B, D-38124, and was assigned the deposit number DSM 28484.

Pichia kluyveri strain No. 2 was deposited at 24.8.2006 by the Okland University Institute of Life Sciences (University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand) of Okland 1142, New Zealand, at the National Institute of measurements Institute of Clark Street No. 541-65, Nanmelbour, 3205, Australia, 541-65 Clarstreet, South Melbourne, Victoria 3205, Australia, and was assigned the deposit number V06/02271.

Pichia kluyveri strain No. 3 was deposited at 24.8.2006 at the national institute for measurements of the city of Okland University, Auckland 1142, New Zealand, by the Okland University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, Okland, N.C. 3205 Nanmelband Clark street 541-65, Victoria, Australia, under deposit number V06/02272.

In another aspect, the invention provides the use of pichia kluyveri to ferment root vegetables. In some embodiments, pichia kluyveri strain 1, pichia kluyveri strain 2, pichia kluyveri strain 3, mutants thereof, or any combination thereof are used to ferment root vegetables to obtain the vegetable juice described herein.

The inventors have demonstrated that pichia kluyveri strains have a great influence on the flavor profile of the final fermented juice product. It has surprisingly been found that for all desired flavour compounds, such as esters and terpenes, a substantial increase is obtained. As shown in the examples, the flavor analysis corresponded well to sensory evaluation. Sensory evaluation showed that the vegetable juice fermented with pichia kluyveri had less fishy off-flavor, and more fruity and spicy aroma than the unfermented control.

The invention also provides products of pichia kluyveri strain 1 in frozen, freeze-dried or liquid form, including for example lyophilized forms and spray/fluidized bed dried forms or frozen or lyophilized concentrates. Different forms of formulations are known in the art and are described, for example, in WO2011/134952(chr. Pichia kluyveri can be grown and concentrated in a fermentor. Additionally, cryoprotectants may be added to maintain viability of the yeast at low temperatures. The product may be added directly to a substrate comprising root vegetables.

Furthermore, the present invention provides a composition comprising pichia kluyveri strain 1 for use in the preparation of a fermented beverage.

Item

The following are preferred embodiments of the invention:

1. the method for reducing the fishy smell and the peculiar smell of the vegetable juice comprises the following steps:

a) providing a substrate comprising a root vegetable,

b) adding at least one Pichia kluyveri (Pichia kluyveri) strain to the substrate,

c) the substrate comprising the pichia kluyveri strain is fermented for less than 48 hours,

d) obtaining the fermented vegetable juice.

2. The method of item 1, wherein the root vegetable is selected from the group consisting of: carrot, beetroot, parsnip, swedish cabbage, turmeric, ginger, potato, sweet potato, turnip cabbage, cassava, cabbage, onion, garlic, celery root, horseradish, white radish, yam bean, radish, jerusalem artichoke, and yam.

3. The method of any one of the preceding items, wherein the pichia kluyveri is selected from the group consisting of:

a) pichia kluyveri, deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ) given deposit number DSM 28484.

b) Pichia kluyveri, deposited at 24.8.2006 at the national institute of measurements and research, 3205 Nanmeuer Clark street 541-65, Victoria, Australia by the University of Okland, Okland 1142, New Zealand, University of Auckland, School of Biological Sciences, Auckland 1142, Okland, New Zealand, in New Zealand, was assigned the deposit number V06/02271.

c) Pichia kluyveri, deposited at 24.8.2006 by the Okland University institute of Life Sciences (University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand) of Okland 1142, New Zealand, at the national institute of measurements and research, No. 541-65, Nanmelban Clark street 3205, Victoria, Australia, under accession numbers V06/02272, and

d) a), b) or c).

4. The process according to any one of the preceding items, wherein the weight ratio of isoamyl acetate to ethyl acetate is at least 0.1, preferably at least 0.2.

5. The method according to any one of the preceding items, wherein step b) further comprises adding a lactic acid bacterial strain to the matrix.

6. The process according to any of the preceding items, wherein the fermentation in step c) is carried out at a temperature of 4-35 ℃, more preferably 18-22 ℃.

7. The method of any of the preceding items, wherein the beta-phenylethyl alcohol in the fermented vegetable juice is at least 0.4 ppm.

8. The method of any one of the preceding items, wherein the alpha-terpineol in the fermented vegetable juice is at least 50 ppb.

9. The method according to any one of the preceding items, wherein the c-whisky lactone in the fermented vegetable juice is at least 4 ppb.

10. The method according to any one of the preceding items, wherein vanillin in the fermented vegetable juice is at least 4 ppb.

11. A fermented vegetable juice obtainable by the method of any one of the preceding items.

12. Fermented vegetable juice comprises root vegetables and Pichia kluyveri.

13. The fermented vegetable juice of claim 12 wherein pichia kluyveri is selected from the group consisting of:

a) pichia kluyveri, deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), assigned deposit number DSM28484, b) Pichia kluyveri, deposited at 24.8.2006 at the national institute for measurements of science of Okland University (University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand) of Okland 1142, Okland, New Zealand, N.3205 south Melbourne street 541-65, Victoria, Australia, assigned deposit number V06/02271,

c) pichia kluyveri, deposited at 24.8.2006 by the Okland University institute of Life Sciences (University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand) of Okland 1142, New Zealand, at the national institute of measurements and research, No. 541-65, Nanmelban Clark street 3205, Victoria, Australia, under accession numbers V06/02272, and

d) a), b) or c).

14. Fermented vegetable juice according to any of claims 12-13 wherein the weight ratio of isoamyl acetate to ethyl acetate is at least 0.1, preferably and/or wherein the weight ratio of isoamyl acetate to ethanol is at least 0.0002, preferably at least 0.0008.

15. The fermented vegetable juice of any one of claims 12-14 wherein the beta-phenylethyl alcohol in the fermented vegetable juice is at least 0.4 ppm.

16. Fermented vegetable juice according to any of claims 12-15 wherein the alpha-terpineol in the fermented vegetable juice is at least 50 ppb.

17. Fermented vegetable juice according to any of claims 12-16 wherein the c-whisky lactone in the fermented vegetable juice is at least 4 ppb.

18. The fermented vegetable juice of any one of claims 12-17 wherein vanillin in the fermented vegetable juice is at least 4 ppb.

19. Application of Pichia kluyveri in fermenting root vegetables is disclosed.

20. The use of item 19, wherein the root vegetable is selected from the group consisting of: carrot, beetroot, parsnip, swedish cabbage, turmeric, ginger, potato, sweet potato, turnip cabbage, cassava, cabbage, onion, garlic, celery root, horseradish, white radish, yam bean, radish, jerusalem artichoke, and yam.

21. The use of claim 19 or 20, wherein the pichia kluyveri is selected from the group consisting of:

a) pichia kluyveri, deposited at the German Collection of microorganisms of Huofen street 7B, D-38124 on 5.3.2014 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), given deposit number DSM28484,

b) pichia kluyveri, deposited at 24.8.2006 at the national institute of measurements and research, No. 541-65, Nanmeuer Clark street 3205, Victoria, Australia by the University of Okland, Okland 1142, New Zealand, of Okland, New Zealand, of New Zealand, with a deposit number V06/02271,

c) pichia kluyveri, deposited at 24.8.2006 by the Okland University institute of Life Sciences (University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand) of Okland 1142, New Zealand, at the national institute of measurements and research, No. 541-65, Nanmelban Clark street 3205, Victoria, Australia, under accession numbers V06/02272, and

d) a), b) or c).

22. Pichia kluyveri, which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ, 7B, D-38124 on 5.3.2014 at Deutsche, D-38124, was assigned the deposit number DSM28484, and a mutant thereof.

23. A composition for fermentation comprising the pichia kluyveri of item 22.

24. The composition of claim 23, wherein the composition is in frozen, lyophilized, or liquid form.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preservation and expert solutions

The applicant requires that prior to the date of patenting, only the deposited microorganism samples described below be provided to the expert.

Pichia kluyveri strain 1 was deposited at DSMZ (German Collection of microorganisms, Deutsche, 7B, D-38124, Brevender Hoffing street) by Chr. Hansen A/S, Denmark at 5.3.2014, and was assigned the deposit number DSM 28484. The deposit is made according to the Budapest treaty on the International recognition of the deposit of microorganisms for the purposes of patent procedure.

Pichia kluyveri strain 2(PK-KR1) and Pichia kluyveri strain 3(PK-KR2) were deposited at 24.8.2006 at national institute of bioscience of Okland University of Okland, Okland 1142, New Zealand, New University of Biological Sciences, Auckland 1142, national institute of measurement and research, No. 541-65, southern Melbourne street 3205, Victoria, Australia, with accession numbers V06/02271 and V06/02272, respectively, as described in WO 2009/110807.

Examples

Example 1

Carrot juice having a pH of 4.5 is provided as a vegetable substrate. It is prepared by diluting a carrot juice concentrate.

A laboratory scale fermentation (200ml) was performed. Carrot juice was extracted with two different Pichia kluyveri strains at a ratio of 10⁵CFU/ml inoculation, i.e., Pichia kluyveri strain 1(DSM 28484) and strain 2 (deposited at the national institute for measurements, accession No. V06/022711). The fermentation was left to stand in an incubator at 20 ℃ for 24 hours. And then sampling.

Ethanol analysis

Ethanol was measured using the ethanol enzymatic bioanalytical kit from Boehringer Mannheim. And (3) fermenting the strain 1 and the strain 2 with the pichia kluyveri for 24 hours to obtain the ethanol concentration of the fermentation product. Table 1 shows the results of the ethanol concentration in the unfermented and fermented carrot juice.

Table 1: ethanol concentration before and after fermentation

Sample (I)	EtOH％(v/v)	EtOH(ppm)
			Not fermented	0.12	946.8
Fermentation with Pichia kluyveri Strain 1	0.34	2682.6
			Fermentation with Pichia kluyveri Strain 2	0.30	2367

As shown, the ethanol concentration in the final fermented carrot product was below 0.05% (v/v).

Flavour analysis

Flavor analysis of all fermentation products was performed in the flavor analysis and enzymology laboratories of salagosa, spain. Tables 2-6 show the concentrations of the different flavor compounds in the samples.

Table 2: concentration of Ethyl acetate before and after fermentation

Sample (I)	Ethyl acetate (ppm)
		Not fermented	0.00
Fermentation with Pichia kluyveri Strain 1	10.30
		Fermentation with Pichia kluyveri Strain 2	25.06

Table 3: acetate (isoamyl acetate and isobutyl acetate) concentrations before and after fermentation

Table 4: ratio of isoamyl acetate/ethyl acetate before and after fermentation

Table 5: ratio of isoamyl acetate/ethanol before and after fermentation

Sample (I)	Ratio of isoamyl acetate (ppm) to ethanol (ppm)
		Not fermented	0.00013
Fermentation with Pichia kluyveri Strain 1	0.00085
		Fermentation with Pichia kluyveri Strain 2	0.00108

Table 6: beta-phenylethyl alcohol concentration before and after fermentation

Sample (I)	Beta-phenylethyl alcohol (ppm)
		Not fermented	0.10
Fermentation with Pichia kluyveri Strain 1	0.86
		Fermentation with Pichia kluyveri Strain 2	0.44

Table 7: alpha-terpineol concentrations before and after fermentation

Sample (I)	Alpha-terpineol (ppb)
		Not fermented	2.67
Fermentation with Pichia kluyveri Strain 1	55.77
		Fermentation with Pichia kluyveri Strain 2	51.52

Table 8: concentrations of aroma compounds (c-whiskey lactone and vanillin) before and after fermentation

Sample (I)	c-whisky lactone (ppb)	Vanillin (ppb)
			Not fermented	0.00	1.24
Fermentation with Pichia kluyveri Strain 1	5.44	4.31
			Fermentation with Pichia kluyveri Strain 2	4.91	4.17

Tables 2-4 clearly show that fermentation of carrot juice with pichia kluyveri enhances the ester profile in the final fermentation product. After only 24 hours of fermentation, the ethyl acetate, isoamyl acetate and isobutyl acetate concentrations in the fermented carrot juice increased at least two-fold.

Pichia kluyveri was shown to increase the ratio of isoamyl acetate (ppm) to ethyl acetate. Pichia kluyveri strain 1 produced less ethyl acetate than Pichia kluyveri strain 2, but the amount of isoamyl acetate was similar. As a result, the ratio of isoamyl acetate/ethyl acetate was higher in Pichia kluyveri strain 1.

In addition, pichia kluyveri was shown to increase the ratio of isoamyl acetate (ppm) to ethanol (ppm). Pichia kluyveri strain 1 showed lower isoamyl acetate (ppm)/ethanol (ppm) compared to Pichia kluyveri strain 2, but the amount of isoamyl acetate was similar.

Tables 6-7 show the concentrations of the two flavor compounds, beta-phenylethyl alcohol and alpha-terpineol, respectively. Both compounds increase significantly in the fermented product and contribute to its floral and spicy taste. The amount of beta-phenylethyl alcohol and alpha-terpineol produced by the pichia kluyveri strain 1 is higher than that produced by the strain 2.

In addition to esters, alcohols and terpenes, the inventors have also found that some lactones and even vanillin are increased, giving the fermentation product a sweeter flavor. Table 8 shows the concentrations of c-whiskey lactone and vanillin.

Example 2

Fresh, non-acidified beetroot juice is provided as a vegetable substrate. It is prepared by pressing beetroot. A laboratory scale fermentation (400ml) was performed. The sugar beet root juice was inoculated with the same pichia kluyveri strains, i.e. pichia kluyveri strain 1 and pichia kluyveri strain 2, as described in example 1. The fermentation was left to stand in an incubator at 20 ℃ for 24 hours. And then sampling.

Sensory analysis

Sensory Evaluation was performed using descriptive analytical techniques according to Lawless et al (Lawless, H.T., and H.Heymann., search Evaluation of Food: Practices and principles, "Food Science series, Chapman and Hall.New York (2010)) to evaluate how Pichia kluyveri promotes flavor.

After fermentation, four apparent attributes of the beetroot fermented juice were determined. These attributes are: earthy, general fruity, spicy and sweet. 9 panelists were selected and a training course with these attributes was performed. In training courses, panelists are trained in identifying the aroma and taste of attributes. This is accomplished by smelling the native fishy character of raw beetroot, the pungent character of pepper, the overall fruity character of tropical fruit juices, and the sweet character of carrot juice.

After the training session, panelists were invited to participate in a fermented juice taste session. Different fermented juices as well as unfermented beetroot juice were assigned to three random panelists and used for each evaluation. The juice was served in a wine glass at about 10 ℃. Panelists scored 4 flavors (aroma and taste combinations) of the juices by scoring (scaling) on a scale of 145mm (0 for absence, 145 for high presence), and their subjective overall liking/acceptance of the juices.

Fig. 1 shows the results of the sensory evaluation. The figure shows how five different attributes of different juices are evaluated on a scale of 0-145: earthy taste, overall fruity taste, pungent taste, sweetness and overall liking.

Fig. 1 shows that all the juices fermented by pichia kluyveri have significantly less earthy taste than the unfermented beetroot juice (the beetroot control in fig. 1). Furthermore, the sweetness level of the fermented juice was very similar to the unfermented beetroot control. They were also considered to be more fruity and spicy than the control.

Pichia kluyveri fermented beetroot juice is generally preferred over unfermented beetroot juice due to reduced fishy off-taste and increased fruit flavor. Pichia kluyveri strain 1 is generally more preferred than pichia kluyveri strain 2, although the fishy off-taste perceived in beetroot juice fermented with pichia kluyveri strain 1 is slightly higher than that of strain 2.

The difference in flavor between the ester and the alpha-terpineol was found to be greatest. Closer observation of acetate concentration revealed that the isoamyl acetate/ethyl acetate ratio (a good fruity determination method) increased in both pichia kluyveri strains. This is in good agreement with sensory evaluations, which show that fermentation of vegetable juice results in a higher score for fruity and pungent taste, and a much lower score for earthy off-flavors, compared to unfermented beetroot juice.

PCT/RO/134 Table