Development and development of refined liquid saccharification enzyme*
Zeng Hui He Xinmin Zhang Xinwu Liu Zhongmin
Abstract According to the characteristics of the fermentation maturation mash of the high-conversion saccharification enzyme, screening and research on production of Based on the characteristics of high conversion saccharase fermentation mash, flocculants and flocculation methods for the production of liquid enzyme were screened and studied, and a set of flocculation process which is unique and suitable for industrialized production was formed. The product was efficiently recovered by adopting advanced plate and frame pre-coating process and ultrafiltration membrane concentration and separation technology, so that the recovery rate of a single tank reached 80%, and the comprehensive average recovery rate reached more than 75%, which was an average increase of 7% compared with that of the traditional salting-out process.
Keywords saccharase, flocculation, recovery rate
Usually, people in the fermentation industry tend to focus on improving the fermentation vigor of strains and neglect to improve the recovery rate of the product, which results in a good yield but not a good harvest. This study adopts a unique flocculation process to produce refined liquid saccharase by plate and frame pre-coating and ultrafiltration membrane concentration method, which improves the product quality grade (from industrial grade to food grade), reduces the pollution of filtrate to the environment, and improves the recovery rate by 7% on average compared with that of the traditional salting-out process, and the comprehensive average recovery rate reaches more than 75%. The technical indexes of the product are in full compliance with the standard of QB1805.2-93 industrial saccharification enzyme preparation, and the preservation rate of enzyme activity in 3 months is more than 95%.
1 Materials and equipment
(1) flocculants: APAM, sodium benzoate, diatomaceous earth, zinc sulfate, yellow blood salt, etc..
(2) Preservatives: calcium acetate, penicillin, potassium sorbate, sodium benzoate and so on.
(3) Flocculation tank: V=20 m3, speed: 50-70 r/min.
(4) Polypropylene plate and frame filter press: 60 m2.
(5) Filter cloth washing machine.
(6) Pre-coating tank: V=3 m3, speed: 80 r/min.
(7) Imported UF809 roll membrane ultrafiltration machine.
(8) Storage tank: V=20 m3.
(9) Finished product processing tank: V=3 m3, rotational speed: 50 r/min.
2 Refined and concentrated liquid saccharification enzyme extraction process research
2.1 Determination of the process route
Determination of the process is the core of this process is the selection of the concentration method and to determine the flocculation process. At present, there are 2 concentration methods for similar products in China, one is to rely on the heat source to evaporate the water in the product; the other is to utilize osmotic membrane ultrafiltration to remove the water in the product. Comparing the above two methods, the former has large investment in equipment and consumes much energy; while the latter has less investment, low energy consumption, simple operation, easy cleaning, low maintenance and replacement costs, and high product yield. Therefore, we chose the advanced osmotic membrane ultrafiltration concentration method. Flocculation process is the most critical step in the extraction process, which directly determines the work of plate and frame filtration and other processes and product quality. In recent years, the research on flocculation process are focused on the selection of flocculant, we selected almost no inorganic metal ions flocculant and high-efficiency filtration methods according to the actual situation.
2.2 Process flow
Enzyme fermentation broth → flocculation → plate and frame press filtration → filtrate
↓ ↓
Cake Ultrafiltration Concentration
↓ ↓
Drying for filler or feed Preservation and standardization
↓
Finished product
2.3 Quality of enzyme fermentation broth
The quality of enzyme fermentation broth directly determines the quality and yield of the product. The indexes we set for the quality of fermentation broth are shown in Table 1.
Table 1 Quality indexes of enzyme fermentation broth
Item Mirror inspection Enzyme activity (u/ml) pH DE value
Indicator Mirror inspection normal
No bacteria >2.5×104 4.2-4.6 <10
If the fermentation broth of individual batch fails to reach the requirements of the above indexes, it will be converted to solid powder production. were changed to the production of solid powder.
2.4 Research on flocculation process
The flocculation process is the most critical step in the production process of refined concentrated liquid saccharase. If the flocculation effect is not good, it will lead to long processing time and increase the possibility of bacterial contamination.
Pre-treatment of fermentation broth using flocculation process can remove the bacteria and other insoluble particles in the fermentation broth, which greatly improves the filtration of the fermentation broth and increases the degree of clarification. The mechanism of flocculation is explained as follows:
(1) The flocculant neutralizes the charge on the surface of the suspended particles (there are also carboxyl groups and amino groups on the surface of the bacterial cells, so they have a charge), which ultimately leads to the flocculation of these particles.
(2) flocculant embedding or adsorption, the bacteria and other insoluble particles mechanically adsorbed and embedded in it.
2.4.1 Comparative test of flocculation mini-trial
Each time, 100 ml of sample was taken and flocculated according to the following flocculation method, and then filtered at atmospheric pressure with a funnel lined with filter paper.
Method 1: Take the original sample and filter it directly through the filter paper.
Method 2: Add water 1/3, bran 1%, diatomaceous earth 2%.
Method 3: Add 1/3 water, APAM appropriate amount.
Method 4: Add 30% to 40% water, add bentonite, diatomaceous earth, sodium benzoate, APAM appropriate amount.
Method 5: add water 1/3, wood chips 1%, add appropriate amount of zinc sulfate, yellow blood salt, APAM. the results are shown in Table 2.
From Table 2, it can be seen that Methods 4 and 5 are the most ideal flocculation method, the filtrate is clear and transparent, the filtration rate is fast, the cake has less water, and it can be used in large-scale production. Method 4 is more in line with food hygiene standards. Methods 1, 2 and 3 have different degrees of problems and should be eliminated.
Table 2 Flocculation methods small test results
Methods Filtration speed (s/10 drops) Filtrate color Filtrate brightness Cake condition Analysis of results
1 40 Dark reddish brown Translucent Brightness Cake is not separated before squeezing, and it becomes a paste. Difficult to filter type, this method is not adopted.
2 14 light yellow, not bright enough, the filter cake before extrusion into a loose shape, good separation. With gauze wrapped filter cake for squeezing, the cake into a block, easy to squeeze dry, less water. Easy to filter type, this method can be adopted to speed up the filtration rate.
3 8 light yellow clear liquid, transparent, no paste on the filter paper before extrusion, the appearance of the degree of separation is not as good as method 2. gauze wrapped filter cake for extrusion, the force is large, more water, the cake into a soft mass. Filtration effect is good, but the cake is difficult to squeeze dry, not loose, cake moisture.
4 6 light yellow clear liquid, transparent and bright, the cake is loose before squeezing, good separation. With gauze wrapped filter cake for extrusion, the cake into a block, easy to squeeze dry, less water. A better filtration method, should be adopted.
5 7 light yellow liquid clear and bright with 4
Better a filtration method, should be adopted.
2.4.2 Industrial production
On the basis of the small test, we used four methods to carry out industrial flocculation and filtration of four batches of fermentation broth (calculated according to the dosage of 6m3 fermentation broth).
Method 1: The fermentation broth was filtered directly without any treatment.
Method 2: Add 1% wood chips and 2% diatomaceous earth and filter.
Method 3: Add 1/3 pure water, 1% wood chips, add appropriate amount of zinc sulfate, yellow blood salt, APAM and then filter.
Method 4: Add 1/3 pure water, 1% diatomaceous earth, appropriate amount of bentonite, sodium benzoate, APAM, add flocculant and stir for 30 min (speed of 50-80 r/min) and then filter. The results are shown in Table 3.
From Table 3, it can be seen that the treatment time of Method 1 and Method 2 is long, and the filtrate is not as clear as the latter two methods, and the water content of the cake is high. The treatment effects of Method 3 and Method 4 are basically the same, but considering the filtrate of Method 3 due to the presence of yellow blood salts, etc.
Table 3 Results of industrialized test of flocculation methods
Item Method 1
9602018 batch Method 2
9602007 batch Method 3
9603001 batch Method 4
Batch 9602025
Enzyme activity in canister (u/ml) 28 466 30 428 29 214 29 018
Observation directly after flocculation Dilute, slightly sticky Dilute, with coarse particles Dilute, with obvious flocculation Same as Method 3
Time of filtrate (h) 22 16 6 5
Filter enzyme activity (u/ml) 22 772 23 646 24 540 24 566
Enzyme activity of filtrate (u/g) 7 759 6 432 5 648 5 489
Moisture of filtrate (%) 62 58 53 52
Color of filtrate Brown-red, brighter Brown-red, brighter Light brown-red, bright Light brown-red, brighter Light brown-red, translucent
Volume of filtrate obtained (m3) 5.2 6.2 5.7 5.7 <
Gained volume of finished product (m3) 1.00 1.10 1.21 1.20
Enzyme activity of finished product (u/ml) 104 170 106 437 105 448 104 766
Organoleptic identification of the finished product Brown, viscous brown, slightly viscous Brown, slightly viscous brown, slightly viscous brown, slightly viscous
compounds, which may have an effect on the quality of the finished product. Thus we chose method 4 for production.
In the experimental study of flocculation treatment and process operation, our experience is:
(1) The use of flocculant effect is related to a variety of factors, the most important of which is the concentration of flocculant, stirring speed and stirring time.
(2) When the amount of flocculant is increased from zero, the amount of suspended particles being flocculated increases accordingly, but beyond a certain concentration, the particles that have been flocculated are dispersed again. Therefore, it is necessary to determine the optimal amount of flocculant through the test.
(3) The added flocculant and the particles in the suspension contact is a prerequisite for the occurrence of flocculation, so it needs to be stirred. However, the floc generated is very fragile, excessive stirring will make the floc crushing tendency is greater than the generation tendency, therefore, and must control the stirring speed and time. We adopted the operation of stirring for 30 min and rotating speed of 50-80 r/min according to the actual production situation, and achieved good flocculation results.
2.5 Plate and frame filtration process operation
Liquid enzyme production, the need for filtrate, so the filtrate turbidity directly affects the quality of the liquid enzyme and ultrafiltration equipment. At the beginning of feeding, because the filter cloth is relatively clean, it should rely on the pressure difference in the tank to feed naturally. If the feed is pressurized at the beginning, it will cause turbidity of the filtrate. Wait for a period of time, when the filtrate is clear and the filtration speed is constant, slowly pressurize the feed. It is especially emphasized here that the discharge port of the receiving tank of the plate and frame should have a device to make the turbid liquid return to the flocculation tank, so that the filtrate can be knocked back in time once it is found to be turbid. After each batch of filtration, the filter cloth should be washed and placed neatly for next time.
Plate and frame filtered out of the cake, each manufacturer should be dealt with according to the actual situation. Our factory also produces solid enzymes, in its production process, need a part of the filler material for standardization, so we will filter cake drying treatment to do filler. The dried material has more than 20,000 units of enzyme activity, so the treatment is more ideal.
2.6 Ultrafiltration concentration process research
Ultrafiltration is a pressurized membrane filtration method, its working principle is under a certain pressure, the large solute molecules blocked on one side of the membrane (to stay in the original solution); and the small solute molecules through to the other side of the membrane, so as to achieve the separation of the purification and concentration of the product purpose. Ultrafiltration is suitable for the extraction, purification and concentration of biomacromolecule fermentation products (such as saccharase, α-amylase). The process has the advantages of low cost, convenient operation, mild conditions, better preservation of enzyme vitality, and high product recovery.
We chose the imported UF809 type rolled ultrafiltration membrane, and according to the product quality and process requirements, the ultrafilter was installed in the form of 3 pcs per group and 3 groups in parallel. The inlet pressure of the ultrafilter is about 4×9.8×104 Pa, the outlet pressure is about 2～4×9.8×104 Pa, and the pressure difference between the inlet and outlet is about 1×9.8×104 Pa. The operating temperature is below 40℃. The concentration of the enzyme solution is determined according to the need, generally about 4 to 5 times. The flow rate of the ultrafiltration solution and enzyme activity were measured regularly during operation to confirm whether the ultrafilter was operating normally. The results of ultrafiltration concentration are shown in Table 4.
Table 4 Statistical results of ultrafiltration concentration
Batch
No. Fermentation broth
Enzyme vitality
(u/ml) Fermentation broth
Liquid volume
(m3) Filtrate
volume
(m3) Ultrafiltration
Time
(h) Ultrafiltration Concentration result on fermentation
Yield
(%)
Finished enzyme
vitality
(u/ml) Finished product
Volume
(m3) Concentration
Multiplication
(v/v) Concentration
Multiplication
(u/u) Enzyme activity
Yield
(%)
9507-01 29 871 8 5.8 2.5 120 301 1.54 3.76 4.00 98 77.5
9507-02 27 232 8 6.0 2.3 110 380 1.50 4.00 4.05 97 76.0
9503-04 28 431 8 6.1 2.2 115 182 1.56 3.80 4.05 97 79.0
9508-10 30 781 8 5.5 2.0 123 124 1.60 3.44 4.00 98 80.0
9509-07 31 232 8 6.0 2.1 128 429 1.50 4.00 4.10 99 77.0
9509-09 30 231 8 5.9 2.0 111 718 1.60 3.70 3.75 98 75.0
From Table 4, it can be seen that the time required for each treatment of about 8t of clear liquid is basically around 2.3 h, and the total yield of the concentrated enzyme is above 75%.
2.7 Preservation experiment of refined liquid enzyme concentrate
Add appropriate amount of preservative to the enzyme concentrate (100,000 u/ml), and store it at room temperature for 3 months, then determine the residual rate of enzyme activity, and the results are shown in Table 5.
Table 5: Preservation Experiment of Refined Concentrate Enzyme Concentrate
Adding amount of preservative(%) Residual Enzyme Activity(%)
No. Mouldy and bad smell after one week
Calcium acetate 0.1, penicillin 1.8 million units/m3 97
Sodium benzoate 0.2, penicillin 1.8 million units/m3 96
Sodium benzoate 0.2, potassium sorbate 0.1 98
From the Table 5, it can be seen that the preservation of enzyme vitality in 3 months are higher than 95%. The preservative composed of potassium sorbate and sodium benzoate, with 98% enzyme activity retention rate, is the most ideal among the above three preservatives and suitable for industrial production.
Author: Zeng Hui, He Xinmin, Zhang Xinwu, Sanmenxia Fermentation Plant, Lushi, 472200
Liu Zhongmin, Institute of Biology, Henan Academy of Sciences, Zhengzhou, 450008
References
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