Chlorella vulgaris intervenes specialty flavor beer and its making process

Abstract

The application discloses a Chlamydomonas reinhardtii intervention characteristic flavor fine-brewed beer and a manufacturing process thereof. The manufacturing process comprises the following steps: preparing a Chlamydomonas reinhardtii glucose suspension through Chlamydomonas reinhardtii; mixing the Chlamydomonas reinhardtii glucose suspension and pure fruit juice and performing pre-fermentation, obtaining a fruit juice fermentation bottom liquid after the pre-fermentation; performing saccharification and preliminary fermentation on saccharification raw materials, obtaining a wort fermentation liquid after the preliminary fermentation; mixing the fruit juice fermentation bottom liquid and the wort fermentation liquid, performing main fermentation, and obtaining a mixed fermentation liquid after the main fermentation; and obtaining the Chlamydomonas reinhardtii intervention characteristic flavor fine-brewed beer after the mixed fermentation liquid is cooled and pressure-prepared and after-ripening of the beer liquid is completed. The Chlamydomonas reinhardtii with high starch content and rich nutrition is used as a carbon source required by fruit pre-fermentation, and the beer yeast is used for fermentation and covering of the characteristic flavor of the fruit, so that the deodorization of the Chlamydomonas reinhardtii is achieved, and the health care function of the Chlamydomonas reinhardtii and the characteristic fruit flavor are introduced into the fine-brewed beer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of beer brewing, in particular to a Chlamydomonas reinhardtii intervention specialty flavor specialty beer and a preparation process thereof. BACKGROUND

[0002] Beer is rich in a large number of amino acids, vitamins, sugars and inorganic salts and other nutrients, and is the third most consumed beverage in the world, only next to water and tea. Craft beer is a product of beer enthusiasts who simply pursue higher beer quality, and there is no clear definition, but it is highlighted that no artificial chemicals are added, and the cost is not considered too much in pursuit of higher quality. The current domestic mass beer market is saturated and the commodity is seriously homogenized, and based on people's pursuit of high-end specialty beer products, fruit-flavored craft beer has also begun to popularize.

[0003] Pre-fermentation of fruits followed by addition of fruit juice to the fermenting wort at different stages of brewing according to the needs of the wine style is one of the common techniques to introduce fruit flavor into beer. This method has the advantages of higher fusion of fruits and beer flavor, easier control of the influence of fruits (such as organic acid components) on the main fermentation of beer, etc. compared with direct addition of fruit juice. However, when pre-fermenting fruits, additional starch hydrolysis emulsion needs to be added to provide carbon source for the growth of yeast used in pre-fermentation unless the fruits themselves contain a large amount of fermentable sugar. Chlamydomonas reinhardtii is an edible green algae, which can be used as a good source of chlorophyll, with a protein content of more than 40%, and is rich in eight essential amino acids and elements such as calcium, magnesium, sodium, potassium, phosphorus, selenium and zinc. The cellulose content is also much higher than that of Spirulina and Chlorella. Chlamydomonas reinhardtii also has the advantages of high starch content, no or only simple breaking wall treatment, etc. Therefore, Chlamydomonas reinhardtii has a wide application prospect as a new food raw material. However, there are few reports on the application of Chlamydomonas reinhardtii in the field of food processing in China, and there is no related beer product. Chlamydomonas reinhardtii has a certain degree of fishy smell as an algae, which is the main reason limiting its use as a food processing raw material. The common methods for removing fishy smell from algae are: using adsorbents such as activated carbon to remove fishy smell, β-cyclodextrin embedding to mask fishy smell, and neutral protease treatment to remove fishy smell. The above methods can reduce the fishy smell to different degrees, but they are not suitable for the production of craft beer because they have a great influence on the quality and flavor of the beer body, and the additional use of artificial agents is contrary to the concept of craft beer. Yeast deodorization technology refers to the conversion of fishy smell components into non-fishy smell substances through the metabolism of yeast. At present, there have been studies on the application of yeast deodorization technology in the deodorization of Chlamydomonas reinhardtii, but the deodorization effect is not good. For example, Zhao Jinyao et al. reported in the Chinese publication "China Food Additives", No. 9, 2021, pp. 44-50 that the deodorization effect of β-cyclodextrin and yeast was compared, and the results showed that only yeast could not remove all the fishy smell, and the flavor was not coordinated.

[0004] Therefore, there is an urgent need to provide a production process of Chlamydomonas reinhardtii specialty flavor craft beer with fruit flavor and no fishy smell. SUMMARY

[0005] The purpose of the present application is to overcome the above technical deficiencies, and to provide a Chlamydomonas reinhardtii intervention specialty flavor craft beer and its production process, which solves the technical problem that the introduction of Chlamydomonas reinhardtii into beer brings fishy smell.

[0006] In a first aspect, the present application provides a production process of Chlamydomonas reinhardtii intervention specialty flavor craft beer, comprising the following steps:

[0007] S1, preparing a Chlamydomonas reinhardtii glucose suspension by Chlamydomonas reinhardtii;

[0008] S2, mixing the Chlamydomonas reinhardtii glucose suspension and pure fruit juice and pre-fermenting, and obtaining a fruit juice fermentation bottom liquid after the pre-fermentation is completed;

[0009] S3, saccharifying and preliminarily fermenting the saccharification raw material, and obtaining a wort fermentation liquid after the preliminary fermentation is completed;

[0010] S4, mixing the fruit juice fermentation bottom liquid and the wort fermentation liquid, and performing main fermentation under the conditions of the preliminary fermentation in S3, and obtaining a mixed fermentation liquid after the main fermentation is completed;

[0011] S5, after the mixed fermentation liquid is cooled and pressurized, the specialty flavor craft beer with Chlamydomonas reinhardtii intervention is obtained after the wine liquid is completed.

[0012] In the second aspect, the application provides a specialty flavor craft beer with Chlamydomonas reinhardtii intervention, which is obtained by the preparation process of the specialty flavor craft beer with Chlamydomonas reinhardtii intervention provided in the first aspect of the application.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] In the application, Chlamydomonas reinhardtii with high starch content and rich in nutrients is used as a carbon source for fruit pre-fermentation, and the masking effect of beer yeast fermentation on the characteristic flavor of fruit is utilized to achieve the purpose of deodorizing Chlamydomonas reinhardtii, so that the health care effect and characteristic fruit flavor of Chlamydomonas reinhardtii are introduced into the craft beer; unlike traditional deodorizing technology, no chemical reagent is added for deodorizing.

[0015] The process of the application introduces Chlamydomonas reinhardtii and fruit juice into the craft beer, which not only adds diversified nutritional ingredients and health care effects to the beer, but also enables the finished beer product to have a unique fruit flavor, which is of great significance for expanding the domestic homogenized beer market and filling the gap in the field of Chlamydomonas reinhardtii food processing, and provides a new idea for the development of specialty flavor craft beer and the application of Chlamydomonas reinhardtii in the field of food processing. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a process flow chart of an embodiment of the preparation process of the specialty flavor craft beer with Chlamydomonas reinhardtii intervention provided by the application. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the application clearer, the application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the application and do not limit the application.

[0018] In a first aspect, the present application provides a process for preparing a Chlamydomonas reinhardtii-intervened specialty-flavor craft beer, comprising the following steps:

[0019] S1, preparing a Chlamydomonas reinhardtii glucose suspension by Chlamydomonas reinhardtii;

[0020] S2, mixing the Chlamydomonas reinhardtii glucose suspension and pure juice and performing pre-fermentation, and obtaining a juice fermentation bottom liquid after the pre-fermentation is completed; the present application introduces fruit flavor into the beer and makes the flavor coordinated by using this step, and the deodorization effect is achieved through the short-time fermentation which is different from the main fermentation of the beer.

[0021] S3, performing saccharification and preliminary fermentation on the saccharification raw material, and obtaining a wort fermentation liquid after the preliminary fermentation is completed;

[0022] S4, mixing the juice fermentation bottom liquid and the wort fermentation liquid, maintaining the preliminary fermentation conditions in S3 to perform main fermentation, and obtaining a mixed fermentation liquid after the main fermentation is completed;

[0023] S5, cooling the mixed fermentation liquid and then pressurizing, and obtaining the Chlamydomonas reinhardtii-intervened specialty-flavor craft beer after the beer liquid is matured.

[0024] The present application firstly adopts the form of co-fermentation of juice and Chlamydomonas reinhardtii to obtain a juice fermentation bottom liquid, and uses the juice fermentation bottom liquid in the subsequent process of preparing a craft beer. The inventors accidentally found that the specialty-flavor craft beer obtained by the present application has a unique fruit flavor and no odor residue, which indicates that the present application has a good deodorization effect by using the masking effect of beer yeast fermentation on the fruit specialty flavor.

[0025] In the embodiment, step S1 specifically comprises:

[0026] S11, culturing Chlamydomonas reinhardtii to a cell concentration reaching a target concentration to obtain a Chlamydomonas reinhardtii cell suspension;

[0027] S12, crushing the Chlamydomonas reinhardtii cell suspension to obtain a Chlamydomonas reinhardtii starch suspension;

[0028] S13, enzymatically degrading the Chlamydomonas reinhardtii starch suspension to obtain a Chlamydomonas reinhardtii glucose suspension.

[0029] Further, in step S11, the target concentration is 10 6 -10 8 / mL, further 10 7 / mL.

[0030] Further, in step S11, the culturing process comprises: inoculating Chlamydomonas reinhardtii into a Chlamydomonas reinhardtii TAP culture medium to perform normal-pressure culture, and performing pure oxygen aeration treatment, and culturing to a cell concentration reaching a target concentration.

[0031] Furthermore, in step S12, the crushing method is low-temperature and high-pressure crushing.

[0032] Furthermore, in step S13, during the enzymatic hydrolysis process, the mixed enzyme used is a mixture of α-amylase and β-amylase, and the mass ratio of α-amylase to β-amylase is 1:(0.5-2), further 1:1; the amount of the mixed enzyme added is 0.2%-0.8% of the mass of the Chlamydomonas reinhardtii starch suspension, further 0.5%; the enzymatic hydrolysis temperature is 20-30°C, further 25°C; the enzymatic hydrolysis time is 20-40min, further 30min.

[0033] Furthermore, after the enzymatic hydrolysis, the method further includes: S14, sterilizing the Chlamydomonas reinhardtii glucose suspension.

[0034] Furthermore, the sterilization method is pasteurization.

[0035] Furthermore, after the sterilization is completed, the method further includes: S15, refrigerating and storing the Chlamydomonas reinhardtii glucose suspension in a sterile environment for later use.

[0036] The present invention does not limit the type of pure fruit juice used in step S2. Those skilled in the art can choose according to actual conditions. In principle, the introduced fruit flavor can coordinate with the flavor of beer such as malt and not be abrupt, and these fruit flavors can be used to mask the fishy smell to a certain extent. For example, the pure fruit juice is at least one of mulberry juice, cherry juice, peach juice and durian juice. Mulberries, cherries and peaches are local specialty germplasm resources in the Han River Basin, and durian has a large audience due to its unique flavor. They can supplement the human body with a variety of nutrients and have also been reported to have various health benefits such as slowing down hair loss, anemia, anti-oxidation and aging, or anti-inflammation. There are related health products on the domestic market, but the types of related health craft beers are few and the promotion is low. The present invention introduces the above-mentioned characteristic fruit flavors (mulberry juice, cherry juice, peach juice and durian juice) into beer. On the one hand, these fruit flavors can be used to mask the fishy smell to a certain extent. On the other hand, the introduced fruit flavors can be coordinated with the malt aroma and other flavors of the beer without being abrupt. At the same time, the present invention can also give the beer health benefits such as antioxidant and supplementation of chlorophyll and a large amount of essential amino acids for the human body.

[0037] In this embodiment, in step S2, the preparation process of the pure juice includes: pre-processing different fruits according to actual needs, and squeezing the pulp obtained after the pre-processing to obtain the pure juice.

[0038] The present invention does not limit the pretreatment process, and those skilled in the art can select it according to actual conditions. For example, pretreatment includes but is not limited to washing, draining, removing stems, peeling, shelling or core removal.

[0039] In the embodiment, in step S2, the volume ratio of Chlamydomonas reinhardtii glucose suspension to pure fruit juice is 1:(0.5-1.5), and further 1:1.

[0040] In the embodiment, in step S2, the pre-fermentation conditions are as follows: the temperature is controlled at 18-22℃, and further 20℃; the amount of oxygen is 5-10ppm at one time, and further 8ppm; the activated Yarrowia lipolytica is inoculated for atmospheric fermentation for 22-26h, and further 24h; and the inoculation amount is 0.05%-0.15% of the total mass of Chlamydomonas reinhardtii glucose suspension and pure fruit juice, and further 0.1%.

[0041] In the embodiment, in step S3, the saccharifying raw material is at least one of barley malt, wheat malt and oat malt.

[0042] Further, the barley malt accounts for 70%-88% of the mass of the saccharifying raw material, and further 80%; the wheat malt accounts for 10%-20% of the mass of the saccharifying raw material, and further 15%; and the oat malt accounts for 1%-10% of the mass of the saccharifying raw material, and further 5%.

[0043] In the embodiment, in step S3, the saccharifying raw material needs to be ground before saccharification. The present application does not limit the grinding method, which can be selected by those skilled in the art according to the actual situation. In principle, the grinding degree is preferably such that the shell of the saccharifying raw material is broken but not crushed, and the contents are pulverized.

[0044] In the embodiment, in step S3, the saccharification process includes: adding pure water to the saccharifying raw material, then performing multi-stage saccharification, sampling for iodine detection after saccharification is completed, and then raising the temperature to 77℃ to completely terminate the enzyme reaction after successfully passing the iodine detection, and then performing solid-liquid separation on the saccharified wort to obtain the wort.

[0045] Further, the mass of the pure water is 2-3 times that of the saccharifying raw material, and further 2.5 times.

[0046] Further, the multi-stage saccharification process is as follows: first maintaining at 44-46℃ for 10-15min, then maintaining at 54-56℃ for 10-15min, and then maintaining at 64-72℃ for 20-25min.

[0047] Further, the multi-stage saccharification process is as follows: first maintaining at 44-46℃ for 10-15min, then maintaining at 54-56℃ for 10-15min, and then maintaining at 64-72℃ for 20-25min.

[0048] Further, the indicator for successful iodine detection is that no color change is observed when a small amount of saccharified wort is dropped into 1-2 drops of 0.01-0.02mol / L iodine solution.

[0049] Further, after the solid-liquid separation, the process further comprises washing the spent grains obtained after the solid-liquid separation, and combining the obtained wash water with the wort for the subsequent preliminary fermentation process. The purpose of this process is to dissolve the lees remaining in the spent grains with hot water, mainly washing the solid part, and incidental washing of the equipment.

[0050] Further, in the washing process, the mass ratio of pure water to saccharifying raw material is 1:(0.5-2), further 1:1; the temperature of the pure water is 70-80℃, further 75℃.

[0051] In the present embodiment, in step S3, the process of the preliminary fermentation comprises: boiling the wort and optionally the wash water, and adding hops at the beginning of the boiling, followed by whirlpooling and sedimentation to remove impurities, and then cooling and performing the preliminary fermentation. The fermentation is performed until the sugar content of the fermentation broth is reduced to below 5°P, to obtain the wort fermentation broth.

[0052] Further, the boiling stage lasts for 0.5-1.5h, further 1h.

[0053] Further, the amount of hops added is 0.1%-0.2% of the total mass of the wort and the wash water, further 0.15%.

[0054] Further, the hops are Hallertau Tradition, with an α-acid content of 6%-7%, further 6.5%.

[0055] Further, the conditions for the preliminary fermentation are: the temperature is 18-20℃, further 19℃; the amount of oxygen supplied at one time is 8-12ppm, further 10ppm; the pressure is normal pressure; the amount of inoculated ale yeast is 10 6 -10 8 million / mL, further 10 7 million / mL.

[0056] In the present embodiment, in step S4, the fruit juice fermentation base liquid accounts for 3%-7% of the total volume of the fruit juice fermentation base liquid and the wort fermentation broth.

[0057] In the present embodiment, in step S4, the end of the main fermentation is marked by the sugar content being reduced to the range of 3.8-5.0°P and remaining unchanged.

[0058] In the present embodiment, in step S5, the mixed fermentation broth is cooled to 4℃.

[0059] In the present embodiment, in step S5, during the pressure preparation process, pure CO2 is filled until the pressure in the tank rises to 20psi.

[0060] In the embodiment, the duration of the pressure preparation process in step S5 is 5-10 days, and further 7 days. In the present application, after the pressure preparation for 5-10 days, the preliminary tasting is performed, and the liquor is clear and has no significant odor, indicating that the liquor is matured.

[0061] In the present application, the fermentation is performed in a closed fermentation tank.

[0062] In the present application, it is necessary to note that 10 6 , 10 7 , 10 8 respectively refer to the order of magnitude of the number of yeast.

[0063] In the second aspect, the present application provides a Chlamydomonas reinhardtii intervention characteristic flavor craft beer, which is obtained by the preparation process of the Chlamydomonas reinhardtii intervention characteristic flavor craft beer provided in the first aspect of the present application.

[0064] Example 1

[0065] A preparation process of a Chlamydomonas reinhardtii intervention mulberry flavor craft beer, comprising the following steps:

[0066] (1) inoculate Chlamydomonas reinhardtii into TAP medium for normal pressure culture, and perform pure oxygen aeration treatment, and culture until the cell concentration is 10 7 million / mL; take the cultured Chlamydomonas reinhardtii cell suspension for low-temperature high-pressure disruption to obtain a Chlamydomonas reinhardtii starch suspension; perform mixed enzyme hydrolysis on the Chlamydomonas reinhardtii starch suspension to prepare a Chlamydomonas reinhardtii glucose suspension, and add alpha-amylase and beta-amylase in a weight ratio of 1:1 at 0.5% of the mass of the Chlamydomonas reinhardtii starch suspension to perform enzyme hydrolysis, the enzyme hydrolysis temperature is 25℃, and the duration is 30 minutes; then perform pasteurization on the Chlamydomonas reinhardtii glucose suspension, and store it in a sterile environment for cold storage after the pasteurization is completed.

[0067] (2) pretreat mulberries, and squeeze the pulp after the pretreatment to obtain pure mulberry juice.

[0068] (3) uniformly mix the Chlamydomonas reinhardtii glucose suspension prepared and sterilized in (1) and the pure mulberry juice obtained in (2) in a volume ratio of 1:1 to obtain a mixed bottom liquid, then pour the mixed bottom liquid into a closed fermentation tank, control the temperature to be 20℃, the one-time oxygen amount is 8ppm, inoculate activated ale yeast to perform normal pressure fermentation for 24h, the inoculation amount is 0.1% of the mass of the mixed bottom liquid, and the mulberry juice fermentation bottom liquid is obtained after the fermentation is completed.

[0069] (4) After the barley malt, wheat malt and oat malt were grinded as the saccharifying raw materials in a mass ratio of 16:3:1, pure water was added and heated to carry out multi-stage saccharification, the added water was 2.5 times the mass of the raw materials, after the saccharification was completed, a sample was taken for iodine test, after the sample successfully passed the iodine test, the temperature was raised to 77℃ to completely terminate the enzyme reaction; the wort was obtained by solid-liquid separation of the saccharified wort, then the wort was washed with pure water at a temperature of 75℃ and an addition amount of 1:1 of the mass of the saccharifying raw materials; the washing water and the wort were collected and then boiled for 1h, Hallertau Tradition hops (α-acid content 6.5%) accounting for 0.15% of the total mass of the wort and the washing water were added at the beginning of the boiling, then the impurities were removed by whirl deposition, and after cooling, the wort was poured into a fermentation tank for fermentation, the specific parameters of the fermentation conditions were as follows: temperature 19℃, one-time oxygen supply amount 10ppm, normal pressure, and the amount of inoculated ale yeast was 10 7

[0070] (5) The mulberry juice fermentation broth obtained in (3) was mixed with the wort fermentation broth obtained in (4), and the fermentation was continued under the conditions in (4) until the sugar content was reduced to 3.8-5.0°P and remained unchanged, at which time the main fermentation was completed, and a mixed fermentation broth was obtained.

[0071] (6) The mixed fermentation broth was cooled to 4℃, and pure CO2 was filled in until the pressure in the tank was raised to 20psi for pressure preparation, which lasted for 7d, and the wine was matured.

[0072] In this embodiment, the pretreatment of mulberries was specifically: water washing, draining, and removing the stems.

[0073] In this embodiment, the multi-stage saccharification procedure was: 45℃ for 15min, 55℃ for 10min, and 70-72℃ for 20min. At this temperature, the activity of α-amylase was relatively higher, and more non-fermentable sugars could be produced, i.e., the sugar content in the finished product was increased, so that the sour and sweet flavors of the mulberry flavor craft beer were highlighted.

[0074] In this embodiment, the added mulberry juice fermentation broth accounted for 7% of the total volume of the juice fermentation broth and the wort fermentation broth.

[0075] Example 2

[0076] A kind of Chlamydomonas reinhardtii intervention's honey peach flavor craft beer making process, comprising the following steps:

[0077] (1) Chlamydomonas reinhardtii was inoculated into TAP medium for normal pressure culture, and pure oxygen was aerated, and the culture was carried out until the cell concentration was 10 7 ​Chlamydomonas reinhardtii cell suspension is obtained by low-temperature high-pressure disruption of the Chlamydomonas reinhardtii cell suspension that has been cultured to completion; the Chlamydomonas reinhardtii starch suspension is subjected to mixed enzyme enzymolysis to prepare a Chlamydomonas reinhardtii glucose suspension; 0.5% of α-amylase and β-amylase by mass of the Chlamydomonas reinhardtii starch suspension are mixed and added for enzymolysis at a temperature of 25°C for 30 minutes; the Chlamydomonas reinhardtii glucose suspension is then subjected to pasteurization, and after sterilization is completed, is stored in a cold storage environment under sterile conditions for later use.

[0078] (2) The honey peaches are pretreated, and the pulp after the pretreatment is completed is juiced to obtain pure honey peach juice.

[0079] (3) The Chlamydomonas reinhardtii glucose suspension prepared and sterilized in (1) and the pure honey peach juice obtained in (2) are uniformly mixed at a volume ratio of 1:1 to obtain a mixed base liquid, and then the mixed base liquid is put into a sealed fermentation tank, the temperature is controlled at 20°C, the one-time oxygen amount is 8 ppm, and activated Saccharomyces eubayanus is inoculated for atmospheric pressure fermentation for 24 h, the inoculation amount is 0.1% of the mass of the mixed base liquid, and after the fermentation is completed, a honey peach juice fermentation base liquid is obtained.

[0080] (4) Barley malt, wheat malt, and oat malt are ground after being taken as saccharification raw materials at a mass ratio of 16:3:1, pure water is added and warmed to perform multi-stage saccharification, the water added is 2.5 times the mass of the raw materials, after the saccharification is completed, a sample is taken for iodine detection, after the iodine detection is successfully passed, the temperature is raised to 77°C to completely terminate the enzyme reaction; the wort is obtained by solid-liquid separation of the saccharified wort, and then the wort is washed using pure water at a temperature of 75°C and an addition amount of 1:1 of the mass of the saccharification raw materials; the washing water and the wort are collected and then boiled for 1 h, Hallertau Tradition hops (α-acid content 6.5%) accounting for 0.15% of the total mass of the wort and the washing water are put in at the beginning of boiling, and then rotary sedimentation is performed to remove impurities, and after cooling, the wort is injected into a fermentation tank for fermentation, the specific parameters of the fermentation conditions are: temperature 19°C, one-time oxygen amount 10 ppm, atmospheric pressure, and Saccharomyces eubayanus inoculation amount 10 7 million / mL of the fermentation liquid; the wort fermentation liquid is obtained when the sugar degree is reduced to below 5°P based on the sugar degree of the fermentation liquid.

[0081] (5) The honey peach juice fermentation base liquid obtained in (3) and the wort fermentation liquid obtained in (4) are mixed, and fermentation is continued under the conditions in (4) to reduce the sugar degree to the range of 3.8-5.0°P and keep it unchanged, at which time the main fermentation is completed, and a mixed fermentation liquid is obtained.

[0082] (6) The mixed fermentation liquid is cooled to 4°C, and at the same time, pure CO2 is filled in until the tank pressure rises to 20 psi for pre-pressurization, and the pre-pressurization is continued for 7 d, and after the wine is matured, the maturation is completed.

[0083] In this embodiment, the pretreatment of the honey peaches is peeling and pitting.

[0084] In this embodiment, the multi-stage saccharification procedure is: 45℃ for 15 min, 55℃ for 10 min, and 64-65℃ for 20 min. At this temperature, the activity of β-amylase is relatively higher, and more fermentable sugars can be produced, i.e., the sugar content in the finished beer is lower, so that the fruity and malty flavors of the honey peach flavored craft beer are highlighted.

[0085] In this embodiment, the added honey peach juice fermentation substrate accounts for 5% of the total volume of the juice fermentation substrate and the wort fermentation liquid.

[0086] Example 3

[0087] A production process of a cherry flavored craft beer with Chlamydomonas reinhardtii intervention, comprising the following steps:

[0088] (1) inoculate Chlamydomonas reinhardtii into TAP medium for atmospheric pressure culture and pure oxygen aeration treatment, and culture until the cell concentration is 10 7 / mL; take the cultured Chlamydomonas reinhardtii cell suspension for low-temperature high-pressure disruption to obtain a Chlamydomonas reinhardtii starch suspension; perform mixed enzyme hydrolysis on the Chlamydomonas reinhardtii starch suspension to prepare a Chlamydomonas reinhardtii glucose suspension; add α-amylase and β-amylase in a weight ratio of 1:1 at 0.5% of the mass of the Chlamydomonas reinhardtii starch suspension for enzyme hydrolysis at a temperature of 25℃ for 30 minutes; then perform pasteurization on the Chlamydomonas reinhardtii glucose suspension, and store it in a sterile environment for cold storage after sterilization.

[0089] (2) pretreat the cherries, and squeeze the pulp after the pretreatment to obtain pure cherry juice.

[0090] (3) uniformly mix the Chlamydomonas reinhardtii glucose suspension prepared and sterilized in (1) with the pure cherry juice obtained in (2) in a volume ratio of 1:1 to obtain a mixed substrate, and then pour the mixed substrate into a sealed fermentation tank, control the temperature at 20℃, control the one-time oxygen amount at 8ppm, inoculate activated AY yeast for atmospheric pressure fermentation for 24h, the inoculation amount is 0.1% of the mass of the mixed substrate, and obtain a cherry juice fermentation substrate after the fermentation is completed.

[0091] (4) Take barley malt, wheat malt and oat malt as saccharifying raw materials in a mass ratio of 16:3:1, grind them after adding pure water and warming them to perform multi-stage saccharification, the added water is 2.5 times the mass of the raw materials, take a sample to perform iodine test after saccharification is completed, and after successfully passing the iodine test, warm them to 77℃ to completely terminate the enzyme reaction; solid-liquid separation is performed on the saccharified wort to obtain wort, then pure water with a temperature of 75℃ and an addition amount of 1:1 of the mass of the saccharifying raw materials is used to wash the grains; the wort and the washing water are collected and mixed, then boiled for 1h, Hallertau Tradition hops (α-acid content 6.5%) accounting for 0.15% of the total mass of the wort and the washing water are put in at the beginning of boiling, then rotary sedimentation is performed to remove impurities, and after cooling, the wort is poured into a fermentation tank for fermentation, the specific parameters of the fermentation conditions are as follows: temperature 19℃, one-time oxygen supply amount 10ppm, normal pressure, and the amount of inoculated ale yeast is 10 7

[0092] (5) The cherry juice fermentation bottom liquid obtained in (3) is mixed with the wort fermentation liquid obtained in (4), and the conditions in (4) are maintained to continue fermentation until the sugar degree is reduced to the range of 3.8-5.0°P and remains unchanged, at which time the main fermentation is completed, and a mixed fermentation liquid is obtained.

[0093] (6) The mixed fermentation liquid is cooled to 4℃, and pure CO2 is filled in until the pressure in the tank is increased to 20psi for pressure preparation, which lasts for 7d, and after aging, the wine is completed.

[0094] In this embodiment, the pretreatment of cherries is specifically: water washing, draining, removing stems, and removing pits.

[0095] In this embodiment, the multi-stage saccharification procedure is: 45℃ for 15min, 55℃ for 10min, and 64-65℃ for 20min. The activity of β-amylase is relatively higher at this temperature, and more fermentable sugars can be produced, that is, the sugar content in the finished wine is lower, so that the fruit aroma and malt aroma of the cherry-flavored craft beer are highlighted.

[0096] In this embodiment, the added cherry juice fermentation bottom liquid accounts for 7% of the total volume of the juice fermentation bottom liquid and the wort fermentation liquid.

[0097] Example 4

[0098] A kind of chlorella intervention durian-flavored craft beer making process, comprising the following steps:

[0099] (1) Chlorella is inoculated into TAP medium for normal pressure culture, and pure oxygen is aerated, and the culture is performed until the cell concentration is 10 7 ​ / mL; taking the cultured Chlamydomonas reinhardtii cell suspension and performing low-temperature and high-pressure crushing to obtain a Chlamydomonas reinhardtii starch suspension; performing mixed enzyme enzymatic hydrolysis on the obtained Chlamydomonas reinhardtii starch suspension to prepare a Chlamydomonas reinhardtii glucose suspension, and adding α-amylase and β-amylase in a weight ratio of 1:1 according to 0.5% of the mass of the Chlamydomonas reinhardtii starch suspension for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 25°C, and the duration is 30 minutes; then the Chlamydomonas reinhardtii glucose suspension is pasteurized, and after sterilization, it is refrigerated and stored in a sterile environment for future use.

[0100] (2) Pre-treating the durian and squeezing the pre-treated pulp to obtain pure durian juice.

[0101] (3) The Chlamydomonas reinhardtii glucose suspension prepared and sterilized in (1) and the pure durian juice obtained in (2) were uniformly mixed in a volume ratio of 1:1 to obtain a mixed base liquid, and then the mixed base liquid was put into a closed fermentation tank, the temperature was controlled at 20°C, the one-time oxygen flow rate was 8ppm, and activated ale yeast was inoculated for normal pressure fermentation for 24 hours, and the inoculation amount was 0.1% of the mass of the mixed base liquid. After the fermentation was completed, the durian juice fermentation base liquid was obtained.

[0102] (4) Barley malt, wheat malt and oat malt were ground in a mass ratio of 16:3:1 as saccharification raw materials, and then purified water was added and the temperature was increased to perform multi-stage saccharification. The amount of water added was 2.5 times the mass of the raw materials. After the saccharification was completed, samples were taken for iodine test. After successfully passing the iodine test, the temperature was increased to 77°C to completely terminate the enzyme reaction. The saccharified mash was subjected to solid-liquid separation to obtain wort, and then purified water with a temperature of 75°C and an addition ratio of 1:1 to the saccharification raw materials was used for washing the mash. The washing mash water was collected and mixed with the wort, and then boiled for 1 hour. At the beginning of boiling, Hallertau Tradition hops (α-acid content of 6.5%) accounting for 0.15% of the total mass of the wort and washing mash water were added, and then vortex precipitation was used to remove impurities. After cooling, the mash was injected into the fermentation tank for fermentation. The specific parameters of the fermentation conditions were: temperature 19°C, one-time oxygen flow rate 10ppm, normal pressure, and the amount of ale yeast inoculated was 10 7 / mL fermentation liquid; the sugar content of the fermentation liquid is used as the judgment basis, and when the sugar content drops below 5°P, the wort fermentation liquid is obtained.

[0103] (5) The durian juice fermentation base liquid obtained in (3) is mixed with the wort fermentation liquid obtained in (4), and the fermentation is continued under the conditions in (4) to reduce the sugar content to the range of 3.8-5.0°P and keep it unchanged. At this time, the main fermentation is completed and a mixed fermentation liquid is obtained.

[0104] (6) The mixed fermentation liquid is cooled to 4°C and pure CO2 is injected into the tank until the pressure in the tank reaches 20 psi for pressure preparation. This process lasts for 7 days to complete the post-aging of the wine.

[0105] In this embodiment, the pre-treatment of durian is specifically shelling and removing the core.

[0106] In this embodiment, the multi-stage saccharification procedure is: 45℃ for 15min, 55℃ for 10min, and 66-68℃ for 20min. The selection of the saccharification temperature ensures the balance of durian flavor, sweet aftertaste and malt aroma in the finished wine.

[0107] In this embodiment, the added durian juice fermentation substrate accounts for 3% of the total volume of the juice fermentation substrate and the wort fermentation liquid.

[0108] Comparative Example 1

[0109] A production process of a craft beer added with Chlamydomonas reinhardtii not subjected to pre-fermentation treatment, comprising the following steps:

[0110] (1) Chlamydomonas reinhardtii is treated in the same manner as step (1) in Example 1 to obtain a sterilized Chlamydomonas reinhardtii glucose suspension.

[0111] (2) The steps of malt treatment, saccharification, solid-liquid separation, boiling, hop addition, spin settling and cooling, and tank fermentation are sequentially performed to obtain a wort fermentation liquid. The condition parameters of the above steps are consistent with those in step (4) in Example 1.

[0112] (3) The Chlamydomonas reinhardtii glucose suspension obtained in (1) is added to the wort fermentation liquid with a sugar content reduced to below 5°P, and the addition amount is 3.5% of the total volume of the fermentation liquid (which is consistent with the proportion of the actual Chlamydomonas reinhardtii glucose suspension in Example 1), and the fermentation is continued under the original conditions. The condition parameters or subsequent operations are consistent with those in step (6) in Example 1, and the finished wine is obtained after the completion of the wine maturation.

[0113] Comparative Example 2

[0114] A production process of a craft beer, comprising the following steps:

[0115] The steps of malt treatment, saccharification, solid-liquid separation, boiling, hop addition, spin settling and cooling, and tank fermentation are sequentially performed, and the wort fermentation liquid is cooled and pressurized after the sugar content is reduced to below 5°P, and the wine maturation is completed. The condition parameters of the above steps are consistent with those in steps (4) and (6) in Example 1.

[0116] The difference between Comparative Example 2 and Example 1 is that no fruit characteristic flavor is introduced, and the difference between Comparative Example 1 and Example 1 is that no Chlamydomonas reinhardtii glucose suspension is added.

[0117] The products of Examples 1-4 and Comparative Examples 1-2 are subjected to taste testing, and the test results are shown in Table 1.

[0118] Table 1: Comparison of taste evaluation of examples and comparative examples based on the method of the present application

[0119]

[0120] From the comparison of Comparative Example 1 and Comparative Example 2, it can be seen that the addition of the untreated Chlamydomonas reinhardtii glucose suspension directly into beer will destroy the harmony of the beer itself, while the beer produced by the co-fermentation of Chlamydomonas reinhardtii and fruit juice in the examples of the present application not only has better taste and flavor, but also has no algae smell. Compared with Comparative Example 2, Examples 1-4 impart a brand-new characteristic flavor to the traditional craft beer on the basis of the original quality.

[0121] The specific embodiments of the present application described above do not constitute a limitation on the scope of protection of the present application. Any various other corresponding changes and modifications made in accordance with the technical concept of the present application shall be included in the scope of protection of the claims of the present application.

Claims

1. A process for producing a special flavor craft beer with the intervention of Chlamydomonas reinhardtii, characterized in that: The following steps are involved: S1. preparing a Chlamydomonas reinhardtii glucose suspension by using Chlamydomonas reinhardtii; S2, mixing the Chlamydomonas reinhardtii glucose suspension and the purified fruit juice and performing pre-fermentation to obtain a fruit juice fermentation base liquid; S3, saccharifying and preliminarily fermenting the saccharification raw materials to obtain wort fermentation liquid; S4, mixing the juice fermentation base liquid and the wort fermentation liquid, maintaining the preliminary fermentation conditions in S3 for main fermentation, to obtain a mixed fermentation liquid; S5, cooling the mixed fermentation liquid and then pressing it in reserve, and after the fermentation liquid is after-ripened, obtaining a special flavor craft beer with Chlamydomonas reinhardtii intervention; Step S1 specifically includes: S11, culturing the Chlamydomonas reinhardtii until the cell concentration reaches a target concentration to obtain a Chlamydomonas reinhardtii cell suspension; S12, crushing the Chlamydomonas reinhardtii cell suspension to obtain a Chlamydomonas reinhardtii starch suspension; S13, enzymatically hydrolyzing the Chlamydomonas reinhardtii starch suspension to obtain a Chlamydomonas reinhardtii glucose suspension; during the enzymatic hydrolysis process, the mixed enzyme used is a mixture of α-amylase and β-amylase; The saccharification raw material is at least one of barley malt, wheat malt and oat malt.

2. The process for producing the special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 1, characterized in that: In step S11, the target concentration is 10 6 -10 8 / mL; In step S12, the crushing method is low-temperature and high-pressure crushing; In step S13, during the enzymatic hydrolysis process, the mass ratio of α-amylase to β-amylase is 1:(0.5-2); the amount of the enzyme mixture added is 0.2%-0.8% of the mass of the Chlamydomonas reinhardtii starch suspension; the enzymatic hydrolysis temperature is 20-30° C.; and the enzymatic hydrolysis time is 20-40 min.

3. The process for producing the special flavor craft beer with the intervention of Chlamydomonas reinhardtii according to claim 2, characterized in that: Step S1 further includes: S14, sterilizing the Chlamydomonas reinhardtii glucose suspension; S15, refrigerate the Chlamydomonas reinhardtii glucose suspension under a sterile environment for later use; wherein, The sterilization method is pasteurization.

4. The process for producing the special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 1, characterized in that: In step S2, the purified juice is at least one of mulberry juice, cherry juice, peach juice, and durian juice; the volume ratio of the Chlamydomonas reinhardtii glucose suspension to the purified juice is 1:(0.5-1.5); and the pre-fermentation conditions are as follows: controlling the temperature at 18-22°C, a one-time oxygen flow rate of 5-10 ppm, inoculating activated ale yeast for normal pressure fermentation for 22-26 hours, and inoculating in an amount of 0.05%-0.15% of the total mass of the Chlamydomonas reinhardtii glucose suspension and the purified juice.

5. The process for producing the special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 1, characterized in that: In step S3, the saccharification process includes: adding purified water to the saccharification raw material, then performing multi-stage saccharification, sampling after the saccharification is completed for iodine testing, heating to 77° C. to completely terminate the enzyme reaction after successfully passing the iodine test, and then performing solid-liquid separation on the saccharified mash to obtain wort; The mass of the purified water is 2-3 times that of the saccharification raw material; the multi-stage saccharification process is: first maintain at 44-46°C for 10-15 minutes, then maintain at 54-56°C for 10-15 minutes, and then maintain at 64-72°C for 20-25 minutes.

6. The process for producing special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 5, characterized in that: After the solid-liquid separation, the method further includes: washing the spent grains obtained after the solid-liquid separation, and combining the obtained spent grain washing water with wort for a subsequent preliminary fermentation process; wherein, during the spent grain washing process, pure water with an addition amount and a mass ratio of saccharification raw materials of 1: (0.5-2) is used for washing the spent grains, and the temperature of the pure water is 70-80°C.

7. The process for producing special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 6, characterized in that: The initial fermentation process includes: boiling the wort, adding hops at the beginning of boiling, then swirling to remove impurities, cooling and then performing initial fermentation, and judging the sugar content of the fermentation liquid, when the sugar content drops below 5°P, obtaining the wort fermentation liquid; wherein, The boiling stage lasts for 0.5-1.5h; The amount of hops added is 0.1%-0.2% of the total mass of the wort and the slop water; The hops are Hallertau Tradition, with an α-acid content of 6%-7%; The conditions for the initial fermentation are: temperature of 18-20°C, oxygen flow of 8-12ppm, pressure of normal pressure, and the amount of ale yeast inoculated with 10 6 -10 8 pieces / mL.

8. The process for producing the special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 1, characterized in that: In step S4, the juice fermentation bottom liquid accounts for 3%-7% of the total volume of the juice fermentation bottom liquid and the wort fermentation liquid; in step S4, the sign of the end of the main fermentation is: the sugar content drops to the range of 3.8-5.0°P and remains unchanged.

9. The process for producing special flavor craft beer with Chlamydomonas reinhardtii as claimed in claim 1, characterized in that: In step S5, the mixed fermentation liquid is cooled to 4°C; during the pressure preparation process, pure CO2 is charged until the pressure in the tank rises to 20 psi. The duration of the pressure preparation process is 5-10 days.

10. A special flavor craft beer with Chlamydomonas reinhardtii, characterized in that: The characteristic flavor craft beer with the intervention of Chlamydomonas reinhardtii is obtained by the production process of the characteristic flavor craft beer with the intervention of Chlamydomonas reinhardtii according to any one of claims 1 to 9.

Images