Hansenula polymorpha strain LM12 and its use in the preparation of coffee with a pineapple flavor

Abstract

The application discloses a strain of cactus Hansenula spodoptera LM12 and application thereof in preparation of pineapple-flavored coffee, the cactus Hansenula spodoptera has a preservation number of CGMCC No.35856 in China General Microbiological Culture Collection Center, the cactus Hansenula spodoptera LM12 has low-temperature resistance, can be applied in preparation of pineapple-flavored coffee at low temperature, and the addition of the cactus Hansenula spodoptera LM12 can effectively promote the pineapple aroma to be integrated into coffee beans, and solves the problem of excessively high product acidity in preparation of the pineapple-flavored coffee, thereby providing a new technical scheme for producing high-quality, non-bad-flavor and aroma-outstanding pineapple-flavored coffee, and the application is suitable for industrialized production and market popularization and application.

Description

Technical Field

[0001] This invention relates to a cactus spore-bearing Hansenula yeast and its application in the processing of pineapple-flavored coffee, belonging to the field of coffee preparation. Background Technology

[0002] In recent years, flavored coffees have continued to gain popularity in the coffee market due to their unique sensory experience and rich product diversity. Among them, fruit-flavored coffees, especially tropical fruit flavors such as pineapple-flavored coffee, have shown great market potential with their fresh, sweet, and complex aroma characteristics. To effectively integrate pineapple flavor into coffee beans, co-fermentation of coffee beans with microbial strains and pineapple juice is a promising technological approach. This method aims to promote the penetration, transformation, and integration of flavor precursors in pineapple juice into the coffee beans through microbial metabolism, thereby obtaining a coffee product with a natural and full-bodied pineapple flavor. However, existing technologies face technical bottlenecks in achieving high-quality pineapple-flavored coffee fermentation: 1. The generation of undesirable flavors (rotten fruit taste): Co-fermentation of pineapple juice and coffee beans at normal ambient temperatures (25-30℃) results in a relatively rapid fermentation process that is difficult to precisely control. This temperature range is highly susceptible to the growth of unwanted microorganisms (such as spoilage bacteria) and accelerates the decomposition of certain components in the coffee beans or pineapple juice. This often results in unpleasant rotten fruit or overripe fermentation flavors in the fermentation products, severely masking the fresh pineapple flavor and significantly reducing the sensory quality and commercial value of the product. 2. Excessive pungent acidity: Pineapple juice itself contains various organic acids (such as citric acid and malic acid), and coffee beans also produce acids during fermentation. Under normal fermentation conditions, the accumulation of these acids often lacks effective control, leading to a high overall acidity and a sharp, pungent fruity acidity in the final pineapple-flavored coffee product. This excessive acidity not only affects the balance of flavors but also suppresses the release and perception of other pleasant aromas (such as the ester aromas of pineapple and the aroma of the coffee itself), resulting in an unharmonious flavor and an underdeveloped aroma.

[0003] Low-temperature fermentation (e.g., 10-15℃) can theoretically slow down the fermentation rate, inhibit the growth of unwanted microorganisms, thereby reducing the generation of undesirable flavors and allowing flavor compounds more time to penetrate into the coffee beans. However, conventional brewing yeasts or commercial starter cultures typically experience a significant decrease in activity or even inactivation at low temperatures, making effective and controllable fermentation impossible at around 10℃, thus hindering the implementation of low-temperature fermentation strategies. Therefore, the industry urgently needs to develop a specialized starter culture that can operate efficiently and stably at low temperatures (e.g., around 10℃). This starter culture must possess the following key characteristics: 1. Excellent low-temperature fermentation tolerance: It must maintain high activity at around 10℃ to ensure the effective fermentation process, extend the fermentation time window to facilitate the full penetration of flavor compounds, and effectively inhibit the growth of unwanted microorganisms, preventing the generation of rotten fruit flavors. 2. Significant acid-reducing properties: It can effectively metabolize or transform excess organic acids accumulated during fermentation (especially acids that produce a pungent taste), significantly reducing the overall acidity of the final product, eliminating pungent fruit acidity, thereby improving the smoothness and harmony of the flavor, and allowing the target pineapple aroma and coffee aroma to be presented more fully and prominently. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a *Hansenula polymorpha* yeast (…). Hanseniaspora opuntiae LM12 was deposited on September 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35856. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0005] Another objective of this invention is to apply the aforementioned cactus spore-bearing Hansenula LM12 in the processing of pineapple-flavored coffee.

[0006] To achieve the above objectives, the present invention adopts the following technical measures:

[0007] 1. Using the pericarps of fresh coffee cherries collected from Baoshan City, Yunnan Province as samples, multiple purified strains were obtained through isolation and screening.

[0008] The strain LM12, which can produce β-D-glucosidase and esterase, was selected through β-D-glucosidase activity assay, esterase activity assay, and low-temperature fermentation experiment.

[0009] 2. Identification of strain LM12

[0010] (1) Morphological characteristics of strain LM12: On YPD solid medium, the colonies are raised, round, white, opaque, and have a dry and smooth surface. Under a microscope, the cells are oval.

[0011] (2) Molecular identification: Genomic DNA of strain LM12 was extracted using a bacterial genomic DNA extraction kit. Using the extracted genome as a template, PCR amplification was performed using ITS universal fungal primers. After sequencing of the PCR products, the sequencing results were compared with the sequences on NCBI. Based on the morphological characteristics and molecular identification results, the strain was finally identified as *Hansenula polymorpha*. Hanseniaspora opuntiae ), named LM12;

[0012] (3) The low-temperature fermentation experiment proved that strain LM12 has low-temperature resistance;

[0013] 3. Applying strain LM12 to the fermentation of pineapple-flavored coffee solved the problems of low-temperature fermentation feasibility and excessively high product acidity, providing a new technical solution for producing high-quality pineapple-flavored coffee with no unpleasant flavors and a prominent aroma.

[0014] Advantages and technical effects of the present invention:

[0015] 1. The strain LM12 provided by this invention has low temperature resistance and can obtain a large amount of mycelium through simple liquid fermentation. The mycelium is easy to obtain, low in cost, and has the potential for commercial application.

[0016] 2. Strain LM12 can be used in the preparation of pineapple-flavored coffee at low temperatures. The addition of Hansenula polymorpha spores LM12 can solve the problem of excessive acidity in the preparation of pineapple-flavored coffee, and provide a new technical solution for producing high-quality pineapple-flavored coffee with no unpleasant flavor and prominent aroma. This invention is suitable for industrial production and market promotion. Attached Figure Description

[0017] Figure 1 The results of β-D-glucosidase production by strain LM12;

[0018] Figure 2 The results of esterase detection for strain LM12;

[0019] Figure 3 The colony morphology of strain LM12 on YPD plates;

[0020] Figure 4 This is a microscopic image of the cell morphology of strain LM12.

[0021] Figure 5 The growth curve of strain LM12 at 10℃;

[0022] Figure 6 This is a schematic diagram of the morphology of pineapple coffee during the fermentation process.

[0023] Figure 7 This is a schematic diagram of the sensory evaluation results. Detailed Implementation

[0024] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Those skilled in the art can implement the invention by referring to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention;

[0025] The aescin screening medium (1L) in the following examples is: aescin 0.3%, ferric citrate 0.05%, NaCl 0.2%, MgSO4·7H2O 0.05%, KH2PO4 0.1%, sterilized at 120℃ for 20 min;

[0026] Esterase screening medium (1L): 1.0% peptone, 0.5% NaCl, 0.01% CaCl2·2H2O, 1.5% agar, pH 6.8, sterilized at 121℃ for 20 min, with the addition of 0.5% sterile Tween 80.

[0027] Example 1: Isolation, screening and identification of Hansenula polymorpha LM12 from cactus

[0028] 1. Samples were taken from fresh coffee cherries collected from Baoshan City, Yunnan Province. The pericarps were added to sterile 0.9% physiological saline, homogenized, diluted, and spread on YPD solid medium. The samples were incubated at 30°C for 48 hours. Single colonies were picked and further isolated and purified by streaking on YPD solid medium to obtain multiple purified strains.

[0029] 2. Strains screening

[0030] A. Production of β-D-glucosidase

[0031] Sterile aescin medium was added to a 96-well plate at a volume of 200 μL, and 20 μL of activated purified bacterial culture was inoculated. The plate was incubated at 28℃ for 48 h. The activity of β-D-glucosidase produced by the test strain was determined according to the color development level: dark black was marked as "+++" indicating the highest enzyme activity, black was marked as "++" indicating the medium enzyme activity, and dark gray was marked as "+" indicating the low enzyme activity.

[0032] Results of β-D-glucosidase production by different strains are shown in the figure. Figure 1 As shown in the figure, strain LM12 produces β-D-glucosidase with an activity of +++. β-D-glucosidase not only has cellulose saccharification activity but is also closely related to terpene aroma precursors. This indicates that strain LM12 has a significant potential for producing aroma metabolites.

[0033] B. Esterase

[0034] The purified bacterial suspension activated with YPD medium was spotted onto esterase screening medium and incubated at 28℃ for 72 h. The formation of a white solid mass around the colony indicated esterase activity. Esterase production results for different strains are shown below. Figure 2 As can be seen from the figure, strain LM12 has a strong ability to produce esterase, and its potential to form esters through enzymatic catalysis is relatively large.

[0035] 3. Identification of strain LM12

[0036] (1) Morphological characteristics of strain LM12: On YPD solid medium, the colonies are raised, round, white, opaque, and have a dry and smooth surface. Under a microscope, the cells are oval. Figure 3 , 4 );

[0037] (2) Molecular identification: Genomic DNA was extracted from strain LM12 using a bacterial genomic DNA extraction kit. Using the extracted genome as a template, PCR amplification was performed using ITS universal fungal primers (TCCGTAGGTGAACCTGCGG, TCCTCGCTTATTGATATGC). After sequencing of the PCR product, the sequencing results are shown in SEQ ID NO:1. The sequencing results were compared with the sequence on NCBI. Based on the morphological characteristics and molecular identification results, the strain was finally identified as *Hansenula polymorpha*. Hanseniaspora opuntiae ), named LM12;

[0038] (3) Low-temperature fermentation characteristics

[0039] The growth curve of strain LM12 cultured in YPD medium at 10°C is shown below. Figure 5 During the lag phase (0-12h), the growth rate is slow; during the logarithmic growth phase (12-36h), the growth rate is at its maximum; after 36h, it enters the stationary phase, with the OD600nm value remaining at approximately 1.06. In production, strains with shorter lag phases have better adaptability, can quickly gain dominance, reduce the formation of byproducts, shorten the fermentation cycle, and thus improve fermentation efficiency. This indicates that yeast LM12 has low-temperature resistance and is suitable for fermenting coffee at low temperatures.

[0040] Example 2: Application of Hansenula polymorpha LM12 in the preparation of pineapple-flavored coffee

[0041] 1. Preparation of fermentation broth of strain LM12

[0042] Activated *Hansenula polymorpha* LM12 was inoculated into YPD medium at an inoculation rate of 1%–3% (v / v) and cultured on a shaker at 200 rpm and 30°C for 18 h. This process was repeated three times to obtain the third-generation fermentation broth. The third-generation fermentation broth was centrifuged at 7000g for 10 min at 4°C, and the precipitate was collected. The precipitate was washed three times with sterile physiological saline, resuspended, and the bacterial density was adjusted to 10⁻⁶. 9 CFU / mL was used to obtain an LM12 bacterial suspension.

[0043] 2. Preparation of pineapple-flavored coffee (see...) Figure 6 )

[0044] (1) Coffee bean selection: The raw material is commercial beans obtained by washing the Yunnan Catimor variety coffee. Broken beans, moldy beans, insect-infested beans, spotted beans, shell beans, shrunken beans and other defective beans and impurities are picked out and used for later use.

[0045] Coffee bean cleaning: Rinse coffee beans 3-5 times with sterile boiling water and soak for 5 minutes to remove surface dust and impurities and reduce microbial contamination. Place them in a pre-sterilized fermentation tank, seal with sterile sealing film, and allow to cool naturally until the temperature drops to 25-35℃.

[0046] Pineapple juice preparation: Cut the pineapple into chunks, juice it using a high-speed blender, and set aside;

[0047] (2) Mix the washed coffee beans with pineapple juice at a mass ratio of 1:1, ensuring the pineapple juice just covers the coffee beans, and add the mixture to the fermentation tank; inoculate the LM12 bacterial suspension from step 1 onto the surface of the coffee beans, achieving a microbial concentration of 10 after inoculation. 6 CFU / g; After inoculation, coffee beans were transferred to a constant temperature chamber, where the temperature was maintained at 10℃ and the relative humidity at 85%, and fermented for 48 hours; From sterilization to the completion of fermentation, the fermentation tank was sealed with a sealing film for microbial culture; the pore size of this sealing film is such that microorganisms cannot pass through, but gas can pass through freely;

[0048] (3) After the fermentation is complete, wash the coffee beans with water 2-3 times to stop the fermentation and remove the pineapple juice. Only keep the coffee beans and dry them at 40-45℃ until the moisture content is 12%. Put the coffee beans into a drum coffee roaster, with the bean temperature at 200℃ and the heat at 50%. The roasting time is 8-12 minutes and the optimal output temperature is 205-218℃. This will give you medium-roasted pineapple-flavored coffee beans (medium to light roast is recommended, with pineapple juice and bacteria).

[0049] Meanwhile, a control group of coffee beans fermented with pineapple juice was set up, using the same method as steps (2) and (3) above, except that strain LM12 (pineapple juice group) was not inoculated; a room temperature group of coffee beans fermented with pineapple juice + bacteria was set up, using the same method as steps (2) and (3), except that the fermentation temperature was 30℃.

[0050] 3. Determination of physicochemical indicators

[0051] (1) Colony count

[0052] The processed green coffee beans were diluted 10-fold with physiological saline solution until a final concentration of 10 was reached. -6 Dilution. Yeast counts were quantified on YPD medium and microbial counts were quantified on PCA (bacterial count) medium. Microbial counts are expressed as logarithmic colony-forming units (cfu / g sample).

[0053] Table 1 shows that sterile boiling water rinsing and soaking effectively removes surface bacteria from raw coffee beans. The microbial count of both the raw coffee beans and the coffee beans with added pineapple juice was <10 CFU / g after 36 hours. The yeast count in the pineapple juice + microbial culture group (10℃) reached 1.2 × 10⁻⁶ after 36 hours. 8 The CFU / g indicates that strain LM12 can grow well in pineapple and coffee bean substrates. At room temperature, the total number of yeasts in the pineapple juice + bacteria (room temperature) group was significantly higher than that in the low temperature group, and the number on PCA plates was significantly higher than that on YPD plates, indicating that in addition to the inoculated yeast strain LM12, room temperature conditions led to the growth of other miscellaneous bacteria.

[0054] Table 1. Microbial count of green coffee beans

[0055] ;

[0056] (2) The protein content was determined in accordance with the national standard GB / T 5009.5-2016 "Determination of protein in food, spectrophotometry"; the total sugar content was determined in accordance with the national standard GB / T 5009.8-2023 "Determination of total sugar in food".

[0057] (3) Determination of total phenol and flavonoid content

[0058] Preparation of coffee extract: Accurately weigh 1g of coffee powder and 15mL of 75℃ deionized water into a 50mL centrifuge tube. Heat in a 75℃ water bath for 5min, centrifuge at 8000 rpm for 5min, and take the supernatant. Filter with filter paper and repeat the above conditions twice.

[0059] Take 500 μL of the processed coffee extract and mix it with 3.5 mL of distilled water in a test tube. Add 250 μL of Folin-Ciocalteu reagent. After standing for 5 minutes, add 1.25 mL of 20% Na2CO3 solution and incubate at room temperature in the dark for 40 minutes. Measure the absorbance at 725 nm and calculate the total phenol content according to the standard curve.

[0060] Weigh 0.2g of sample powder, add 25mL of methanol, weigh, reflux in a 75℃ water bath for 1h, add weight after cooling, and filter; take 0.2mL of filtrate, dilute to 10mL with methanol, and measure the absorbance at a wavelength of 283nm. Calculate the total flavonoid content according to the standard curve.

[0061] (4) Determination of chlorogenic acid, caffeine and trigonelline content

[0062] UPLC was used to determine the contents of chlorogenic acid, caffeine, and trigonelline in green coffee beans from different treatment groups. A stock solution of 1 mg / mL standard was prepared using methanol. Standard solutions of 10 μg / mL, 20 μg / mL, 40 μg / mL, 100 μg / mL, and 200 μg / mL were prepared, and a standard curve was plotted. 0.5 g of sample was weighed, added to 50 mL of 70% methanol solution, and extracted by sonication for 30 min. The sample was then centrifuged at 5000 rpm for 10 min, and the supernatant was collected for analysis.

[0063] Table 2 Nutritional components of green coffee beans

[0064]

[0065] Different letters in the same row indicate significant differences (P<0.05).

[0066] Table 2 shows that under low-temperature fermentation conditions, compared to Catimor commercial beans, the total sugar, protein, and chlorogenic acid contents of coffee beans in the pineapple juice group and the pineapple juice + yeast group did not change significantly. However, the total phenolic and total flavonoid contents of coffee beans in the pineapple juice + yeast group were significantly higher, both exceeding those in the pineapple juice group and Catimor commercial beans. Although pineapple juice itself contains certain polyphenols and flavonoids, the addition of *Hansenula polymorpha* LM12 can decompose and metabolize green coffee beans and pineapple juice, producing additional polyphenols and flavonoids. Furthermore, the caffeine and trigonelline contents were significantly reduced after processing, which to some extent reduced the bitterness of the coffee, consistent with the sensory evaluation results. Compared to fermentation at 10℃, room-temperature fermentation with pineapple juice + yeast showed stronger yeast metabolic activity and decomposition and metabolism between raw material components, resulting in a significant decrease in total sugar and total protein contents and a significant increase in total phenolic contents in the coffee beans.

[0067] (5) Determination of organic acids

[0068] Organic acids were detected using high-performance liquid chromatography (HPLC). Mobile phase A was 0.5% potassium dihydrogen phosphate solution (pH adjusted to 2.4 with phosphoric acid), mobile phase B was methanol, the volume ratio of A to B was 97:3, the injection volume was 10 μL, the column temperature was 35℃, isocratic elution was set for 10 min, the flow rate was set to 0.7 mL / min, and the detection wavelength was set to 210 nm.

[0069] Table 3 Organic Acid Content of Green Coffee Beans

[0070]

[0071] Different letters in the same row indicate significant differences (P<0.05).

[0072] Coffee beans contain various organic acids, which give coffee its unique acidity, such as citric acid and berry acid. The type and content of organic acids are one of the important factors determining coffee quality. The main organic acids affecting coffee acidity include citric acid, malic acid, and quinic acid. Increasing their concentration has a positive impact on the acidity, berry flavor, and fruit flavor in coffee. Citric acid mainly produces acidity in coffee, while malic acid mainly produces astringency. A moderate increase in organic acids can make coffee flavor more pleasant, but excessive concentrations can easily lead to a bitter and astringent taste and poor flavor. Compared to commercial beans, coffee treated with pineapple juice shows a significant increase in citric acid, quinic acid, and malic acid, while the content of oxalic acid and acetic acid is low and does not change significantly, which brings a prominent acidity to the coffee beverage. In particular, coffee fermented with both cactus yeast and pineapple juice has a lower organic acid content than coffee infused with pineapple juice alone, indicating that microorganisms can utilize and decompose these organic acids, transforming them into other flavor compounds, resulting in a milder acidity. Overall, coffee beans treated with pineapple juice generally show an increase in organic acid content, resulting in a higher acidity level, consistent with sensory evaluations. Adding yeast for fermentation is a safe and effective method for reducing acidity, effectively lowering the main acids in coffee beans while also positively impacting the final flavor profile. The organic acid content of pineapple juice fermentation at room temperature did not show a significant change compared to fermentation at 10°C.

[0073] (6) Sensory evaluation

[0074] Following the SCA (Specialty Coffee Association) cupping system, sensory analysis was conducted on roasted coffee processed using four different methods. 16.5g of freshly ground coffee powder was accurately weighed and poured into 300mL of 93℃ hot water. The prepared sample liquid (20mL) was then placed in a cupping tasting cup (20℃) for aroma testing. A panel of ten trained judges evaluated the samples according to the SCA cupping protocol. This evaluation considered ten attributes, scoring dry / wet aroma, finish, acidity, body, balance, overall impression, cleanliness, sweetness, and consistency. Consistency, cleanliness, and sweetness were given full marks and not reflected in the evaluation. Sensory indicators were evaluated on a scale of 6 to 10. The sum of the scores for all ten sensory indicators was the final score for each sample.

[0075] The sensory evaluation team members cupped and scored the roasted beans in the following groups according to the SCA international cupping standards: Catimor commercial beans, pineapple juice group, pineapple juice + spawn (10℃) group, and pineapple juice + spawn (room temperature) group. The results are as follows: Figure 7 As shown, the Catimor commercial beans, pineapple juice group, pineapple + mushroom (10℃) group, and pineapple juice + mushroom (room temperature) group received different scores on each sensory attribute, with total scores of 79.96, 80.52, 84.03, and 79.51 respectively. The pineapple + mushroom (10℃) group performed well in flavor, aftertaste, acidity, and balance, and also received a good overall evaluation and total score. The Catimor commercial beans are processed using the traditional washed method. While the roasted beans showed good cleanliness during cupping, their acidity, aroma, and flavor were bland. The pineapple group showed improved dry and wet aroma, flavor, and body compared to the commercial beans, but exhibited sharp acidity and fruitiness, with excessive acidity and weak aroma, resulting in lower acidity and balance scores. However, the addition of pineapple juice and yeast for co-fermentation enhanced the coffee's aroma and softened the acidity, significantly improving its acidity and flavor scores. It imparted tropical fruit aromas such as pineapple, described in cupping as: pineapple aroma, sweetness, nutty notes, tropical fruit flavor, and bright acidity. Compared to the 10°C low-temperature processing, the pineapple coffee fermented at room temperature received a lower overall rating. Its pineapple flavor was not prominent, and it exhibited over-fermented acidity and a rotten taste, resulting in a lower sensory score than the commercial beans.

[0076] (7) Determination of volatile substances

[0077] Qualitative and quantitative analysis of volatile components: The NIST 14.0 mass spectrometry database was used for matching searches of each component. Compounds with a matching degree greater than 80% were selected, while substances lost through column leaching, such as siloxanes, and septum leaching were excluded. Quantitative analysis was performed using the internal standard method with 3-heptanone as an internal standard.

[0078] OAV calculation: OAV is the ratio of the semi-quantitative analysis result of a single volatile compound to the sensing threshold of that substance in water. Generally, it is believed that an OAV greater than or equal to 1 for an aromatic active compound makes a significant contribution to the aroma, and the degree of contribution is directly proportional to its OAV value.

[0079] The content of volatile compounds does not directly reflect their contribution to the overall flavor. Odor threshold is an important characteristic of aroma substances, and the contribution of a single volatile compound to the overall flavor is positively correlated with its OAV. Based on the quantitative analysis results of volatile compounds by GC-MS, and referring to relevant literature on the odor threshold of corresponding substances, OAV was calculated. A total of 22 volatile compounds with OA≥1 were obtained from the three samples, mainly pyrazines, aldehydes, ketones, and esters. As shown in Table 4, 2-ethyl-5-methylpyrazine, methyl-2-pyrrolecarboxaldehyde, β-damascone, and methyl decanoate were the four compounds with the largest OAV, indicating that they make important contributions to the overall aroma of fermented pineapple-flavored coffee, imparting aroma characteristics such as fruitiness, sweetness, roasted nuts, and pineapple. Studies have shown that esters are the main contributors to the pineapple aroma. In this study, the total concentration of volatile esters was as follows: pineapple juice + bacteria (10℃) group (18.73 μg / g) > pineapple juice group (16.74 μg / g) > pineapple juice + bacteria (room temperature) group (15.93 μg / g) > Catimor commercial beans (15.06 μg / g). Methyl decanoate primarily exhibited a pineapple flavor, with the highest concentration in the pineapple and bacteria co-fermentation group, indicating that fermentation effectively promoted the penetration and retention of the pineapple aroma. Methyl-2-pyrrolecarboxaldehyde, β-damascone, 2-acetylpyridine, tea pyrrole, and vanillin were only detected in coffee co-fermented with yeast and pineapple juice, suggesting that *Hansenula polymorpha* spores presents more complex aroma compounds through metabolites or the decomposition of coffee beans and pineapple components. Meanwhile, compared to commercial Catimor beans, the pineapple juice group and the pineapple juice + fermentation group showed significantly lower levels of 2-hydroxy-5-methylacetophenone, indicating that processing effectively reduced the herbaceous and strawy flavors of Catimor beans, which are generally considered undesirable. In the pineapple juice + fermentation (room temperature) group, the levels of isobutyraldehyde and 2-methylquinoxaline were higher, which is due to over-fermentation of the pineapple juice at room temperature, resulting in musty and other off-flavors.

[0080] Table 4. Volatile compounds with OAV > 1 in roasted coffee

[0081] ;

[0082] .

Claims

1. A type of cactus spore-forming Hansenula yeast ( Hanseniaspora opuntiae LM12, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 35856.

2. The application of the cactus spore-forming Hansenula zymophila LM12 as described in claim 1 in the processing of pineapple-flavored coffee.

Images