Test Report on Mixed Fermentation of Wheat Flour and Rice Flour（October 2020）

1.Test purpose

1) To study the effect of mixed fermentation of wheat flour and rice flour.

2) Study the addition and optimization of enzyme preparations for mixed fermentation of wheat flour and rice flour.

2. Raw materials and instruments

Raw materials: commercially available wheat flour, rice flour-crushing workshop.

Excipients: Genencor amylase, Longkote glucoamylase, Xiongjin viscidase, Le Yeast compound enzyme preparation, silver sino viscidase, urea, Anjuntai, Angel dry yeast

Instruments: rapid moisture titrator, constant temperature water bath, stirrer, acidity meter, analytical balance, electronic balance, temperature-controlled electric furnace, viscometer, shaker, volumetric flask, triangular flask, etc.

3. Test content

3.1The content of the first stage experiment

3.1.1. Liquefaction test: to explore the influence of different wheat addition ratios on the liquefaction effect;

3.1.2. Fermentation test: to explore the effects of different wheat additions and enzyme preparations on fermentation;

3.1.3 Waste mash filtration experiment: to explore the effect of enzyme preparation on the filtration rate of wheat blended rice fermentation waste mash.

3.2 The content of the second stage experiment

3.2.1 Evaluation of the fermentation effect of the enzyme preparations of Leye, Xiongjin and Baiyinsai Nuo on wheat mixed rice.

4. experimental methods

4.1(The first stage experimental method）

4.1.1、Configure 28% powder slurry: Weigh the required wheat flour, rice flour, and water according to the test plan, mix them in the liquefaction reactor, and stir evenly;

4.1.2、Adjust the pH value: adjust the pH value of the slurry to 5.4-5.6 with 65% sulfuric acid, add amylase (amylase addition method and addition amount are added according to the test plan in Table 1 and 2), and stir evenly;

4.1.3、Liquefaction: Turn on the water bath 1 hour in advance, and heat the water to 92°C; put the liquefaction reactor into the water bath, install a stirrer, and adjust the speed to 260rpm. When the slurry temperature reaches 85°C, time to liquefy for 2.0h, Detect iodine test and viscosity change.

4.1.4、Fermentation: After the liquefaction, the temperature of the liquefied mash is cooled to room temperature with ice water, and water is added; then the pH value of the liquefied mash is adjusted to about 4.4 and divided into 500mL Erlenmeyer flasks; then various auxiliary materials (addition method and addition amount) Add according to the test plan in Table 1 and 2) Shake well, seal with eight layers of gauze, and weigh; after culturing at 30°C at 120 rpm for 8 hours, adjust the temperature to 33°C and ferment to 70 hours.

4.1.5、Detection: 72 weighing, observing the CO 2 weight loss, after 72 hours of fermentation, testing the viscosity, reducing sugar, residual total sugar, filtered total sugar, wine, volatile acid, acidity, waste mash (80 mesh filter) and the number of yeasts after fermentation. , Germination rate and mortality.

4.1.6、Filtration speed experiment method: the waste mash obtained after the mature mash is distilled to remove the liquor is filtered with an 80-mesh filter cloth, and the time used to filter 5ml, 10ml, 15ml, and 20ml filtrate is recorded respectively.

Table 1 Test plan

Remarks: Addition of auxiliary materials: urea 1.10kg/t cassava, Longkot glucoamylase 0.68kg/t powder, Anjuntai 5ppm; the pre-fermentation is operated in triangular flasks, 2-3 batches per plan, 2 parallel for each gradient ；

Table 2 Test plan

Remarks: Several enzyme preparations are calculated according to the level of enzyme activity to determine the specific addition amount.

4.2（The second phase experiment method）

4.2.1Particle size distribution:

Aged wheat is crushed with 1.8mm and 2.0mm screens, and the particle size distribution experiment is performed with 10, 25, 30, 45, 60, 80, and 100 mesh screens.

4.2.2liquefaction

Take rice flour and wheat, test the moisture content, prepare a flour slurry with a solid content of 28%, and prepare the slurry water according to Table 1. Add 4.8kg/t raw ammonia water, measure pH, add 0.10kg/t amylase powder, weigh, put into 87℃ water bath to liquefy, start timing when the material temperature reaches 87℃, liquefy for 2.5h, start stirring during liquefaction 300r/min. Cool after liquefaction, replenish water, detect solid content, iodine test, viscosity change.

Table 3 Mixing water mixing ratio preparation table

4.2.3Fermentation

Adjust the pH of the liquefied mash to 4.40 with sulfuric acid, and then dispense it into 500ml Erlenmeyer flasks, 400 grams per bottle, add auxiliary materials according to Table 2, seal with gauze, weigh, and incubate at 120r/min in a 30℃ incubator for 8h, then adjust Let stand and ferment for 72h at 33°C. At the end of fermentation, the viscosity, reducing sugar, residual total sugar, filtered total sugar, wine, volatile acid, acidity, waste mash (80 mesh filter), yeast number, germination rate and death rate are detected at the end of fermentation.

Table 4 Fermentation auxiliary material addition table

The added amount of Jituibao 5ppm yeast is 1‰ liquefied mash

3、Experimental method of filtration speed: The waste liquor obtained after the liquor separation was removed by distillation of mature mash was filtered by funnel with 80-mesh filter cloth, and the time for filtering 5ml, 10ml, 15ml and 20ml filtrate was recorded respectively.

5、Test results and analysis

5.1、（First-phase experimental method）

5.1.1. Fermentation effect of different proportions of wheat and rice blended

5.1.1.1Liquefaction test results

Table 5 Liquefaction test results

Liquefaction target control DE value is generally in the range of 12-20%, the smaller the viscosity, the better. It can be seen from Table 5 that as the mixing ratio of wheat increases, the viscosity has a tendency to increase. To a certain extent, the increase in viscosity is related to the content and activity of amylase. When the content of amylase is low, the continuous increase of the substrate concentration leads to the weakening of the ability to hydrolyze starch, and the molecular weight of starch does not decrease, resulting in high viscosity. Studies have shown that the level of protein content directly affects viscosity. In the experiment, the protein content of wheat flour is 12.75% and rice flour protein is 11.2%. When the mixing ratio of raw materials is changed, the protein content in the flour slurry will gradually increase. Acid protease should be appropriately added during the fermentation process. It can improve the fermentation effect.

5.1.1.2Fermentation test results

Table 6 Chemical test results

Table 7 Liquid phase test results

Remarks: Wheat starch 57%

From the chemical test, the acidity and volatile acid are within the normal range, and the fermentation test is normal. From the analysis of wine, with the increase of solid content, the higher the fermented wine. When the rice flour and wheat flour are mixed and fermented, when the solid content is 30%, the 15% wine content of the wheat flour blended reaches the highest, but the residual sugar is relatively high. When the solid content is 29%, with the increase of the blending ratio of wheat flour, the alcohol content does not change significantly, and the residual reducing sugar and the residual total sugar increase. The average value of the liquid phase detection in Table 7 shows that when the proportion of wheat is blended with 15%, the wine is the highest. As the amount of wheat is continuously added, the arabinose content also increases.

5.1.2.Effect of Different Enzyme Preparations on Fermentation Effect of Wheat Blended with Rice

The test results in Table 8 show that there is no doubt that the enzyme preparation directly affects the fermentation effect, which is mainly reflected in the difference in the conversion of starch into yeast available sugar by amylase and saccharification enzyme. The data shows that the effect of adding 20% of wheat to fermentation is better. Under the action of several enzyme preparations, with the addition of the amount of wheat, the amount of residual reducing sugar and residual total sugar also increase, indicating that there may be a certain amount of reducing sugar A large amount of non-fermentable sugar leads to an increase in residual sugar content. Among several enzyme preparations, the use of silver sino enzyme preparation has a slight advantage in improving wine, but the residual reducing sugar is too high.

Table 8 Chemical test results

Remarks: The solid content after liquefaction is 30%

5.1.3.Experimental results of the filtration rate of waste mash

5.1.3.1Effect of different ratios of wheat mixed with rice fermentation mash on filtration rate

Table 9 Experimental results of filtration speed

From Table 9, the filtration speed of the waste mash without adding wheat flour is slower than that of the production rice fermentation waste mash. On the one hand, the reason is that the residual sugar affects the viscosity, which causes the mash to have a certain influence on the filtration speed. After the rice flour is mixed with wheat at 10%, 15%, and 20%, it is difficult to filter the waste mash. The main reason is that the waste mash has a high viscosity (due to the small test test, the sample size is limited and the specific viscosity value is not measured). From the appearance of the mash, the surface layer of the waste mash becomes gelatinous after standing for a certain period of time at room temperature.

5.1.3.2Effect of Enzyme Preparation on the Filtration Rate of Fermented Sap from Wheat Blended with Rice

After several enzyme preparations are used in combination with their viscosity-reducing enzymes/complex enzymes, the filtration rate of waste mash has been greatly improved. When 20% wheat is added, the filtration rate of waste mash is the best. The data shows that the effect of using Xiongjin enzyme preparation is significant in solid-liquid separation, followed by Baiyin Sano.

Table 10 Filtration experiment results

5.2、（The second phase experiment method）

5.2.1.Wheat flour particle size distribution results

Table 11 Particle size distribution of wheat flour

Summary: In the case of a 1.8mm screen, 37.39% is on the 45-mesh sieve and 16.91% is on the 60-mesh sieve. With a 2.0mm screen, 47.23% is on the 45-mesh sieve and 15.83% is on the 60-mesh sieve. Explain that the range of 20-60 mesh accounts for more than 50%.

5.2.2Liquefaction and fermentation test results

The fermentation effect of Le Yeh, Xiongjin and Baiyin Sai Nuo enzyme preparations on wheat mixed rice is shown in Table 3.

Table 12 Chemical test results

3.Experimental results of the filtration rate of waste mash

Table 13 Experimental results of filtration rate

Summary: Judging from the comprehensive analysis of chemical test results and filtration speed: when the addition amount of Le Yeh compound enzyme preparation is 100ppm/t mash, the liquor content is 12.55% (V/V). Compared with other enzyme preparations, the enzyme addition amount is 100ppm/t mash filtrate and it takes only 30 seconds to collect 5ml of mash filtrate.

6、in conclusion

1.When using Genencor amylase and Longcott glucoamylase for mixed fermentation of wheat flour and rice flour, as the mixing ratio of wheat increases, the viscosity tends to increase, and it is difficult to filter the waste mash. When the solid content is 30%, the blending of 15% wheat flour with 15% alcohol will reach the maximum, and the residual sugar will be relatively high. The average value of the liquid phase test shows that as the amount of wheat is continuously added, the arabinose content also increases.

2．Several enzyme preparations combined with their viscosity reducing enzymes/complex enzymes after fermentation data show that the effect of fermentation is better when wheat is added at a ratio of 20%, and the filtration rate of waste mash is greatly improved, so that the waste mash can be separated from the solid-liquid smoothly. However, with the addition of the amount of wheat, the amount of residual reducing sugars and total residual sugars are also increasing, indicating that there may be a certain amount of non-fermentable sugars in the reducing sugars, resulting in an increase in the content of residual sugars.

3．On the basis of the first phase of the experiment, a 30% wheat blending experiment was carried out to evaluate the fermentation effect of the enzyme preparations of Le Yeh, Xiongjin, and Baiyin Sai Nuo on wheat mixed rice. The experiment showed that the addition amount of Le Yeh compound enzyme preparations was used. When it is 100ppm/t mash, the liquor content is 12.55% (V/V). Compared with other enzyme preparations, the enzyme addition amount is 100ppm/t mash filtrate and it takes only 30 seconds to collect 5ml of mash filtrate.