CN107841420A - A kind of method that head mold brews chestnut fruit wine with saccharomyces cerevisiae mixing one-step fermentation - Google Patents

Abstract

The invention provides a method for fermenting chestnut fruit wine by mixing rhizopus and saccharomyces cerevisiae in one step. The method directly uses microorganisms to control the whole process of chestnut wine fermentation, and uses rhizopus instead of amylase to complete saccharification. At the same time, the addition amount and ratio of Rhizopus and Saccharomyces cerevisiae are precisely controlled. Compared with the traditional brewing process of first saccharification and then fermentation, Rhizopus and Saccharomyces cerevisiae are directly added to complete the entire saccharification and fermentation process in one step. It not only simplifies the process steps, saves the cost, but also improves the utilization rate of raw materials. Moreover, the aroma of the chestnut fruit wine is stronger and the taste is better, which can meet the current consumption demand of people for alcoholic drinks.

Description

A kind of method that Rhizopus and Saccharomyces cerevisiae mix one-step fermentation to brew chestnut fruit wine

technical field

The invention relates to the field of wine making, in particular to a method for brewing chestnut fruit wine by mixing Rhizopus and Saccharomyces cerevisiae through one-step fermentation.

Background technique

The existing method of brewing chestnut fruit wine mainly uses amylase, pectinase, glucoamylase or other types of enzymes to saccharify chestnuts, and then inoculates saccharomyces to carry out alcoholic fermentation.

The starch content and properties of chestnut are quite different from those of glutinous rice and sorghum. The starch content in chestnut is relatively high. Studies have shown that southern chestnut also has the characteristics of high amylose content and low peak viscosity. In addition, chestnut fruit is also rich in protein, fat, vitamins, carotene, and minerals such as Ca, Fe, and Sn; traditional distiller's koji has complex types of microorganisms, and fermentation is difficult to control. Commercially available koji is mainly aimed at the fermentation of rice or glutinous rice. The raw materials used for fermentation are different, and the required strains and strain ratios are also different. There is also no special koji for brewing chestnut fruit wine on the market, and the result of making chestnut fruit wine with commercially available koji is unsuccessful.

In the related report of existing brew chestnut fruit wine, must adjust the pH of fermented liquid before fermentation (because the optimum pH of α-amylase is 5.6-5.8, and the optimum pH of β-amylase is 5.4-5.5, fermented liquid be adjusted to weak acidity, which can promote the action of amylase); in the existing reports of brewing chestnut fruit wine, amylase is first used to complete the saccharification process; and southern chestnut has the characteristics of high amylose content; The process causes starch retrogradation, which leads to poor fermentation.

Contents of the invention

The invention provides a method for mixing rhizopus and Saccharomyces cerevisiae to ferment and brew chestnut fruit wine in one step. The method adopts the method of directly using microorganisms to control the whole process of fermentation of chestnut fruit wine, uses rhizopus to replace amylase to complete the saccharification process, and Make full use of the metabolic intermediates produced during the fermentation process of rhizopus to improve the flavor of fruit wine, and at the same time precisely control the amount and ratio of rhizopus and Saccharomyces cerevisiae.

Concrete technical scheme of the present invention is as follows:

A method for fermenting chestnut fruit wine by mixing rhizopus and Saccharomyces cerevisiae in one step, characterized in that: the steps are as follows:

(1) After the chestnuts are cooked and cooled, shelled and peeled, the chestnut kernels are prepared according to the mass-volume ratio:

Distilled water = 1g: 2-8ml, mix with water and mash to obtain chestnut homogenate;

(2) adding edible white granulated sugar to the chestnut homogenate to adjust the sugar content so that the sugar content is 8%-22%;

(3) After sealing the chestnut homogenate obtained in the previous step, sterilize it for 20 minutes at a temperature of 121° C. and a steam pressure of 1 kg/cm ² ;

(4) When the temperature of the sealed chestnut homogenate obtained in the previous step drops to 30° C., inoculate the Rhizopus spore suspension and Saccharomyces cerevisiae, place in a 28-36° C. constant temperature incubator and seal and ferment for 2-8 days to obtain a fermented liquid; The volume ratio of the rhizopus spore suspension and Saccharomyces cerevisiae is 1:0.5-3, and the total inoculation amount of the rhizopus spore suspension and Saccharomyces cerevisiae is 8%-22% of the total volume of chestnut homogenate;

(5) Suction filtration and pasteurization of the fermented liquid obtained in the previous step to obtain the finished chestnut fruit wine.

As a preferred item: the method of cooking and cooling the chestnuts in the step (1) is as follows: after the fresh chestnuts are washed, they are boiled in water, the amount of water is just enough to cover the chestnuts, and then the cooked chestnuts are placed in normal temperature water, cool down.

As a preference item: the preparation method of Saccharomyces cerevisiae in the step (4) is as follows: activate Saccharomyces cerevisiae strains in YPD liquid medium, place the temperature at 28°C and 150rpm shaker to cultivate to the logarithmic growth phase, take the bacterial liquid in Centrifuge at 4°C for 10-15 minutes, take the cell pellet, rinse with sterile water and resuspend to obtain the seed solution.

As a preference item: the preparation method of Rhizopus in described step (4) is as follows: Cultivate Rhizopus 70-72h in the PDA solid slant, get mass volume concentration and be 8.5mg/1mL sterilized physiological saline, add to the root of cultivating Stir and filter with sterilized filter paper to adjust the concentration of spore suspension to 10 ⁶ /mL.

As a preferred item: the mass volume ratio of chestnut kernels and distilled water in the step (1) is 1g:5mL.

As a preferred item: the sugar content in the step (2) is 10%.

As a preferred item: the volume ratio of the rhizopus spore suspension and Saccharomyces cerevisiae in the step (4) is 1:1; the total inoculation amount of the rhizopus and Saccharomyces cerevisiae is 16% of the total volume of the chestnut homogenate.

As a preference: the temperature of the constant temperature incubator in the step (4) is 30°C;

As a preferred item: said step (4) sealed culture for 6 days.

Chestnut (scientific name: Castanea mollissima) in the present invention, has another name called chestnut, Chinese chestnut, chestnut, wind chestnut, is the plant of Fagaceae chestnut; Chestnut is nutritious, and vitamin C content is also higher than tomato, more than ten times of apple; The minerals in chestnuts are also very comprehensive, including potassium, zinc, iron, etc., which are much higher than ordinary fruits such as apples, especially the potassium content is 3 times higher than that of apples; It is rich in starch, protein, fat, calcium, phosphorus, iron and vitamins A, B, C and other substances, especially rich in vitamins B1 and B2, of which the B2 content is 4 times that of rice, and the protein content is 30% higher than that of rice. , the fat is 20 times higher than that of rice; although the Dabie Mountains are rich in chestnuts, the development of chestnut by-products is still insufficient, and the intensive processing lacks technical support. On the one hand, the simple way of raw sales is cheap and cannot provide higher value of chestnuts; on the other hand, chestnuts are not resistant to storage and are extremely perishable, resulting in waste; the development of chestnut fruit wine has solved the above problems well and greatly improved the quality of chestnuts. It improves the added value of chestnut products, improves the technological level of the chestnut industry, and provides a good industrialization prospect for the development of the chestnut industry.

Beneficial technical effects: 1. There is a synergistic effect between Rhizopus and Saccharomyces cerevisiae during the fermentation process. Compared with the simple fermentation of Saccharomyces cerevisiae, the product has a stronger aroma and better taste, which can meet the current consumption needs of people for alcoholic drinks; 2. Compared with amylase, rhizopus can produce organic acids such as fumaric acid and lactic acid while producing amylase; the acid produced by rhizopus just provides a good weak acid environment for amylase; In fruit wine, it is necessary to use chemicals to adjust the acidity of the fermentation broth, which simplifies the fermentation steps and improves the production efficiency; Rhizopus mainly plays a role in saccharification during wine making, and Rhizopus can produce abundant saccharifying amylase, which can convert α in the starch structure -1,4 bond and α-1,6 bond are cut off, so that most of the starch is converted into fermentable sugar. In addition, because Rhizopus contains alcoholic enzymes, it can ferment while saccharifying, so that the utilization rate of starch is improved. Using Rhizopus to complete the saccharification process has higher efficiency and stronger utilization rate, and the intermediate metabolites greatly improve the yield of fruit wine. 3. By using the mixed fermentation of Rhizopus and Saccharomyces cerevisiae, the inoculation amount and fermentation ratio of Rhizopus and Saccharomyces cerevisiae can be precisely controlled, and their energy efficiency and role in different stages of fermentation can be explored, which not only saves costs, but also improves The utilization rate of raw materials also makes the flavor of the product better. In this experiment, rhizopus and Saccharomyces cerevisiae were used for mixed fermentation, and the biologically active enzymes and organic acids produced by the rapid reproduction of rhizopus were used to inhibit the retrogradation of starch and accelerate saccharification, so as to ensure the smooth progress of fermentation; the brewing method of chestnut fruit wine was the same as that reported Compared with this method, this method can effectively inhibit starch retrogradation and accelerate saccharification, and enhance the flavor of fruit wine, which is an unexplored content in the prior art; (4) in the present invention, rhizopus and Saccharomyces cerevisiae are used to mix one-step synergistic fermentation to make chestnut fruit wine, and The rhizopus can saccharify while fermenting, which simplifies the production process of saccharifying first and then fermenting in the prior art, and improves the utilization rate of starch.

Instructions attached

Fig. 1 is a flowchart of the present invention;

Figure 2 is the growth curve of Saccharomyces cerevisiae.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

1. Selection and cooking of chestnuts

The chestnuts used in the experiment were fresh or refrigerated chestnuts stored in the Dabie Mountains. Take out the chestnuts (full and not broken) from the refrigerator, wash them with room temperature water, put 400g of chestnuts in a 1000ml beaker, and add water until the chestnuts are just completely covered. Seal it with plastic wrap to reduce the degree of browning, put it in the microwave oven, adjust the "medium-high heat" and cook for 30 minutes.

2. Shelling and beating

Put the cooked chestnuts in room temperature water, take a cold water bath for 30 minutes, wait for the chestnuts to cool down, remove the shells and skins, take chestnut kernels (m/g):distilled water (v/ml) at a ratio of 1:5, and place them in a tissue grinder Medium, pound for 2 minutes.

3. Sugar adjustment

Adjust the sugar level with the help of a sugar meter, and adjust the sugar level to 10% with edible white sugar.

4. Bottling and sterilization

Take a 250ml Erlenmeyer flask, fill it with 100mL of chestnut homogenate, and steam sterilize it at 121°C and 1kg/ ^cm2 pressure for 20min.

5. Treatment of strains

5.1 Saccharomyces cerevisiae

Activate the Saccharomyces cerevisiae strain in YPD liquid medium, culture it on a shaker at a temperature of 28°C and 150rpm, and cultivate it to the logarithmic growth phase. Take the bacterial liquid and centrifuge it in a centrifuge at 4°C at 4000rpm for 10min, take the cell pellet, and use a sterile After washing with water, resuspend the seed solution.

5.2 Rhizopus

The preparation of rhizopus spore suspension: put rhizopus in PDA solid slant 28 ℃ static culture 3 days, get 10mL concentration and be 0.85% sterilized physiological saline, join in the cultured rhizopus slant culture medium, stir and sterilize Filter with bacterial filter paper, count with a hemocytometer, and adjust the concentration of the spore suspension to 10 ⁶ /mL, and the inoculation amount is calculated by volume.

6. Preparation and cultivation of fermentation broth

Put the sterilized and sealed chestnut homogenate in the ultra-clean workbench, wait for the temperature to drop to 30°C, inoculate 10% of Rhizopus spore suspension + 10% of Saccharomyces cerevisiae according to the inoculation amount (volume percentage), and place at 30°C In a water-proof constant temperature incubator, the culture was sealed for 6 days.

7. Post-processing

The fermented chestnut fruit wine was filtered with a 1mL suction filter bottle by means of a Bush funnel and a vacuum pump;

Sterilized by pasteurization;

Detection of total bacterial count, Escherichia coli and pathogenic bacteria;

Determination of alcohol content, total sugar content and pH determination;

Sealed and preserved.

Exploration of the production process (Note: All the experimental data below are the average value of three experiments) 1. Control of water-material ratio

Make sure that 100ml of chestnut homogenate is installed in each 250ml triangular bottle, control the ratio of water to material 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, and measure it after the finished product is obtained. Physical and chemical indicators, and sensory testing to determine the optimum water-material ratio.

The influence of the control of material-water ratio on the quality of chestnut fruit wine:

It can be seen from Table 1 that if the ratio of material to water is too small, the concentration of each substance in the reaction system will be very large, which will inhibit the progress of the reaction; if the ratio of material to water is too large, the concentration of each substance in the reaction system will become smaller, resulting in the inability of the maximum reaction , and the alcohol content of the finished product is diluted; when the material-water ratio is controlled to be 1:5, the finished product has the highest alcohol content and the highest sensory score, so it can be seen that the optimum material-water ratio is 1:5.

Table 1 Effect of material-water ratio on fruit wine fermentation

2. Control of sugar content before fermentation

The sugar content of chestnut homogenate directly determines the alcohol content of the fermentation broth. After the chestnut is processed into pulp, the measured sugar content is 4%, and the sugar content is adjusted to 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, and the test contains The relationship between sugar content and alcohol content, after the finished product is obtained, its physical and chemical indicators are measured, and a sensory test is carried out to determine the optimum fermentation sugar level.

Effect of added sugar content on the quality of chestnut fruit wine:

Through experiments, it is found that the greater the sugar content of chestnut homogenate, the greater the sugar content of the final fermentation broth. When the sugar content of chestnut homogenate is between 4% and 16%, the alcohol content of the fermentation broth has been greatly increased. , but when the sugar content of the chestnut homogenate reaches 18%, the alcohol content of the fermented liquid no longer increases. It can be seen that the sugar content in the reaction solution is too high, and the excess sugar cannot be transformed. Combined with the sensory evaluation, the optimum sugar content of the chestnut homogenate is obtained 10% (Table 2).

Table 2 Effect of sugar content on the quality of chestnut fruit wine

3. Control of the amount of inoculation

A larger inoculation amount can speed up the fermentation process and improve production efficiency. In the experiment, the volume ratio of Rhizopus and Saccharomyces cerevisiae was inoculated at 1:1, and the total inoculation volume was controlled to be 8%, 10%, 12%, 14%, 16%, 18%, 20%, and 22%. Physical and chemical indicators, and sensory testing to determine the optimum inoculum volume.

Effect of total inoculum amount on the quality of chestnut fruit wine:

The speed of chestnut homogenate fermentation mainly depends on the volume of fermentation broth and the amount of fermentation agent added. On the basis of keeping the volume of fermentation broth constant, controlling the total inoculation amount of bacteria can improve the reaction speed of fermentation broth. Experiments show that the inoculation amount has relatively little influence on sugar content and pH value, but has a greater impact on alcohol content and sensory evaluation. The sugar content is maintained at a level of 7%-9%, while the pH is maintained at 3.57-4.35. When the inoculation amount is 8%, the pH is the highest, and when the inoculum amount is 16%, the pH is the lowest. When the inoculum amount is between 8%-16% When the alcohol content and the sensory score of the finished product are higher, the quality of the finished product is declining when the inoculum amount is greater than 16%. The comprehensive alcohol content and the sensory evaluation determine that the optimum inoculum amount of the bacterial classification is 16% (Table 3).

Table 3 Effect of total inoculum amount on the quality of chestnut fruit wine

4, the control of rhizopus and saccharomyces cerevisiae inoculum ratio

In the whole process of fermentation, Rhizopus is mainly responsible for saccharifying starch and producing flavor substances. Saccharomyces cerevisiae mainly converts sugars into alcohol, but the two interact with each other in the fermentation process. Explore the inoculation ratio of Rhizopus and Saccharomyces cerevisiae , is of great significance. In the process, the total inoculation amount is constant, and the rhizopus is controlled: Saccharomyces cerevisiae=1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1:0.5, to explore the influence of the strain ratio on the experimental results, and obtain The physical and chemical indicators of the finished product are measured, and a sensory test is carried out to determine the most suitable bacterial inoculation ratio.

The influence of the optimum strain ratio on the fermentation of fruit wine:

In the overall process of fermentation, Rhizopus is mainly responsible for saccharifying starch and producing flavor substances, and Saccharomyces cerevisiae is mainly responsible for producing alcohol, but the reaction processes of the two are also complementary and affect each other. By experiment, we can find out that when rhizopus and saccharomyces cerevisiae addition is 1:1, we can obtain the highest alcoholic strength, the best sensory score, because reaction process is very complicated, reason still needs further investigation (table 4 ).

Table 4 The effect of the most strain ratio on fruit wine fermentation

5. Control of fermentation temperature

Temperature also has a great influence on the fermentation process. In the experiment, the fermentation temperature was controlled at 28°C, 30°C, 32°C, 34°C and 36°C, and the physical and chemical indicators of the finished product were measured, and the sensory test was carried out to determine the optimum fermentation temperature.

Optimization of fermentation temperature:

Appropriate fermentation temperature provides a good environment for the fermentation process. When the temperature is between 28°C and 32°C, the quality of fruit wine has been improving, but when the temperature exceeds 32°C, the alcohol content decreases and the sensory score also decreases. It also shows that the temperature is too high to be unfavorable for the fermentation. Combined with the sensory evaluation, we can draw that the optimum fermentation temperature is 30°C (Table 5).

Table 5 Effect of fermentation temperature on the quality of chestnut fruit wine

6. Control of fermentation days

In the experiment, the fermentation days were controlled as 2d, 3d, 4d, 5d, 6d, 7d, and 8d. After the finished product was obtained, its physical and chemical indicators were measured, and a sensory test was carried out. Determine the optimum fermentation days.

Effect of fermentation time on the quality of fruit wine

It can be seen from Table 6 that the alcohol content of fruit wine also rises first and then decreases with the number of days of fermentation, and reaches the maximum alcohol content on the fifth day. However, through sensory testing and comprehensive testing of various aspects, it is concluded that the quality of fruit wine is the best when it is fermented for 6 days Therefore, it is determined that the best fermentation days of chestnut fruit wine are 6 days.

Table 6 Effect of fermentation time on the quality of chestnut fruit wine

7. Orthogonal experiment

Select three single factors that have a great influence on the results, namely sugar adjustment degree, inoculum size and fermentation temperature, and use the L9(34) orthogonal table to conduct orthogonal experiments.

Orthogonal experiment to determine the best fermentation conditions

According to variance analysis, the primary and secondary factors affecting product quality are obtained: sugar adjustment>inoculation amount>fermentation temperature.

8. Sensory evaluation

The sensory evaluation of color, aroma and taste is carried out on the finished product. The evaluation personnel are fixed students from 10 related majors. The sensory evaluation table is shown in the table below:

It should be understood that the parts not described in detail in this specification belong to the prior art.

It should be understood that the above-mentioned descriptions for the preferred embodiments are relatively detailed, and should not therefore be considered as limiting the scope of the patent protection of the present invention. Within the scope of protection, replacements or modifications can also be made, all of which fall within the protection scope of the present invention, and the scope of protection of the present invention should be based on the appended claims.

Claims (9)

1. a kind of method that head mold brews chestnut fruit wine with saccharomyces cerevisiae mixing one-step fermentation, it is characterised in that：Step is as follows：

(1) by after the cooked cooling of Chinese chestnut, shell, remove the peel, be Chinese chestnut Seed according to mass volume ratio：Distilled water=1g：2-8ml ratio Example plus water mixing, smash to pieces, obtain Chinese chestnut homogenate；

(2) edible white granulated sugar is added in Chinese chestnut homogenate and adjusts pol, it is 8% -22% to make its sugar content；

(3) by Chinese chestnut homogenate sealing obtained by upper step after temperature be 121 DEG C, steam pressure 1kg/cm²Under conditions of sterilize 20min；

(4) when the Chinese chestnut homogenized temperature of sealing obtained by upper step is down to 30 DEG C, head mold spore suspension and saccharomyces cerevisiae is inoculated with, is placed in It is sealed by fermentation 2-8 days in 28-36 DEG C of constant incubators, obtains zymotic fluid；The head mold spore suspension and saccharomyces cerevisiae volume Than for 1：0.5-3, total inoculum concentration of the head mold spore suspension and saccharomyces cerevisiae is homogenized 8%-the 22% of cumulative volume for Chinese chestnut；

(5) zymotic fluid obtained by upper step is filtered, sterilized with pasteurization, get product chestnut fruit wine.

2. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Chinese chestnut is cooked cold in the step (1) But method is as follows：After fresh Chinese chestnut is cleaned, it is placed in water cooked, the amount of water there was not Chinese chestnut just, then by well-done Chinese chestnut It is placed in normal-temperature water, cools down.

3. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Saccharomyces cerevisiae in the step (4) Preparation method is as follows：Wine brewing yeast strain is activated in YPD fluid nutrient mediums, is placed in 28 DEG C of temperature, 150rpm shaking table cultures To exponential phase, take bacterium solution to centrifuge 10-15min under the conditions of 4 DEG C, take cell precipitation, must be planted with being resuspended after aseptic water washing Sub- liquid.

4. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：The preparation of head mold in the step (4) Method is as follows：Head mold is cultivated into 70-72h in PDA solid slope culture mediums, takes mass-volume concentration to be sterilized for 8.5mg/1mL Physiological saline, it is added in cultured head mold slant medium, stirs and filtered with sterilizing filter paper, adjust the dense of spore suspension Spend for 10⁶Individual/mL.

5. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Chinese chestnut Seed is with steaming in the step (1) The mass volume ratio of distilled water is 1g：5ml.

6. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Sugar content is in the step (2) 10%.

7. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Step (4) the head mold spore suspension It is 1 with saccharomyces cerevisiae volume ratio：1；Total inoculum concentration of the head mold and saccharomyces cerevisiae is that Chinese chestnut is homogenized the 16% of cumulative volume.

8. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Constant incubator in the step (4) Temperature is 30 DEG C.

9. the method for chestnut fruit wine is brewed as claimed in claim 1, it is characterised in that：Step (4) the sealing culture 6 days.