5. Controles
5.2. Controles relacionados con la confidencialidad
Saccharification of distillery mashes is a somewhat controversial subject. Over the last 10 years many distillers have changed from saccharifying mash in a dedicated sacchari- fication vessel (or ‘sacc’ tank) to adding the saccharifying enzyme directly to the fermentor in a process referred to as Simultaneous Saccharification and Fermentation (SSF). Saccharification in a separate vessel is still the practice in some distilleries, particularly for beverage production and continuous fermentation.
Another factor is use of Rhizozyme™, an enzyme produced by surface culture (solid state fermentation). Rhizozyme™ has an optimum pH of 3.5-5.0 and an optimum temperature of 30- 35°C. As such, it is a glucoamylase more suited
100 75 50 25 0 49 54 60 65 71 77 82 88 93 % activity remaining Temperature (°C)
Substrate: 4% w/v soluble starch + 10 ppm calcium Treatment time: 15 minutes
100
Figure 14. Activity of high temperature-tolerant α-amylase
in relation to temperature.
to a distillery fermentor (Table 5). Rhizozyme™ has particularly found favor in SSF situations because the enzyme can work at near its optimum in the fermentor. Because the organism
used to produce RhizozymeTM is grown on a
solid substrate (eg wheat bran), this enzyme source also contains side activities that assist in releasing more carbohydrate and protein (Table 5). These side activities work to release sugars from other structural carbohydrates besides starch, which also releases bound starch that is then available for conversion. This approach to enzyme addition is able to increase yield to higher levels than normally seen with single activity liquid glucoamylase. Levels in excess of 2.9 gallons/bushel have been reported; and the target is 3.1 gallons/bushel (122 gallons or 456 liters per tonne).
Table 5. Advantages of Rhizozyme™ over conventional glucoamylase. Conventional Rhizozyme™ glucoamylase Temperature optimum, °C 60+ 30+ pH optimum 4.0-5.5 3.5-5.0 Amylase activity, SKB units None 50,000 Cellulase activity
CMC-ase units None 2500
Amylopectinase, AP units None 5000
IF A SACCHARIFICATION STEP IS USED
Mash from the liquefaction vessel is cooled, usually to 60-65°C, and transferred to a saccharification vessel where the glucoamylase (amyloglucosidase) enzyme is added. This exoenzyme starts hydrolyzing the dextrins from the non-reducing end of the molecule and progressively, though slowly compared to endoenzymes, releases glucose. The saccharifying process is usually carried out with a residence time of between 45 and 90 minutes, but can be as long as 6 hrs, and the glucoamylase is added at 0.06-0.08% by weight of cereal used. Some distillers measure the quantity of glucose produced by measuring the dextrose equivalent (DE) of the mash. A DE of 100 represents pure glucose, while zero represents the absence of glucose. This test is rarely used nowadays, as many distillers have high performance liquid
Grain dry milling and cooking procedures 21
chromatography systems (HPLC) that can measure sugars directly. Recent experience, however, shows that DE, provided it is above 10, is of no concern. In focusing on 23% ethanol, the key in any cooking and liquefaction process is to liquefy, i.e., lower viscosity, so that mash can be pumped through the heat exchanger to the fermentor (for SSF) or to the saccharification tank.
The functional characteristics of liquid glucoamylase prepared from the microorganism
Aspergillus niger, can be seen in Figures 15 and
16. Two parameters, temperature and pH, dictate how enzymes can be used. While liquefaction is carried out at a pH of 6.0-6.5 and a temperature of 90°C, this is not at all acceptable for saccharification. The pH must be in the 4-5 range for saccharification; and the optimum temperature for the glucoamylase activity is 60°C. The mash, therefore, must be acidified with either sulfuric acid or backset stillage or both before addition of the glucoamylase. Temperature must also be adjusted. As mentioned previously, normal mash saccharification temperature is 60-65°C; although for microbiological reasons 70-75°C would be preferable. Lactobacillus can survive at 60°C; and frequent infection of saccharification systems has caused many distillers to change to saccharifying in the fermentor.
IF NO SACCHARIFICATION STEP IS USED
If no saccharification step is planned, the liquefied mash is simply cooled from 90°C through a heat exchanger and transferred to the fermentor. A portion of the liquefied mash is diverted to a yeast starter tank where yeast,
glucoamylase and the RhizozymeTM is added.
Conventional glucoamylase (L400) is added at 0.06% with 0.01% Rhizozyme™ recommended as a supplement. Rhizozyme™ alone can be added at 0.05%, in which case no glucoamylase is required.
Recommendations
Given the goals and the variables involved, which cooking system should be chosen? A comparison of systems used in four distilleries
demonstrates the diversity of approach possible, yet points out many similarities (Table 6). It would appear that there are three schools of thought:
a) A long liquefaction period (1-2 hrs at 90+ °C) after the high temperature jet cooker and low temperature slurry (no enzymes).
b) A short high temperature slurry prior to a long liquefaction period (1-2 hrs at 90+ °C) after the high temperature jet cooker. Enzymes used in slurry.
c) Choice of saccharification stage or SSF. From the experience of the authors, recommendations are for a short slurry at 140- 150°F followed by jet cooking (10-15 min.) and a long (1.5 hrs) liquefaction step. The mash should have a sugar profile similar to Table 7. It should then be cooled to fermentation temperature on the way to the fermentor. This profile will ensure a rapid start by yeast with no sugar overload. In the presence of Rhizozyme™ or glucoamylase, dextrins will continue to be ‘spoon-fed’ to the yeast during fermentation. Enzyme additions should include all the amylase during liquefaction and Rhizozyme™ during fermentation.
Table 7. The target sugar profile following liquefaction.
pH 4.2 Solids 29-35 Glucose, % 1.8-2.5 Maltose, % 0.7-0.9 Maltotriose, % 0.2-0.3 Lactic, % 0.07-0.08 Glycerol, % 0.2-0.3
The future
Many new enzyme systems including xylanases, hemicellulases, ligninases (esterases) and others are being developed and the cooking system must be flexible enough to handle them. The objective is to maximize the biochemical activity of the enzyme so as to maximize alcohol yield, not to fit an enzyme to an existing engineering design.