Research and Optimization of Technological Process Based on Fermentation for Production of Seaweed Feed ()
1. Introduction
The seaweed waste, the insoluble fiber residue of which glue, iodine and other substance has been extracted from seaweed waste, mostly used as waste discharged into the natural world directly, that not only caused waste of resources, also resulted in environmental pollution [1], while there are abundant nutrients in the seaweed waste, as high-quality animal feed ingredients, which can effectively improve the immunity of animals and increase the economic benefits [2,3] .
Microbial fermentation which is of advantage with short cycle and high efficiency, has been widely used in food, feed and other industries using the characteristics of the microorganism itself contains large amounts of protein, has a promising prospect. Algae residue contains major required nutrients of animal feed: protein, crude fiber and other trace elements, which the protein and crude fiber content is usually 8.86% - 8.92% and 43.16% - 43.65% respectively [4]. Not only the protein content of algae residue can be increased by using microbial anaerobic fermentation with adding appropriate source of nitrogen and other growth factors, can also make the crude fiber degrade into some monosaccharides and polysaccharides prebiotics which are necessary to animals. This method can effectively improve the nutrient content of the algae residue and enhance its application in animal feed value with the major effects: 1) improve the rumen environment and promote the ecological balance of microorganisms in vivo; 2) improve feed conversion rate; 3) produce organic acids [5,6] . Also this method is of low cost of raw materials, simple process and short cycle, and has not been reported before.
Fermentation results mainly influenced by the active of the bacteria agents, while the activity of the bacteria agents are influenced by the environment and dosage of auxiliaries, these factors impact on the fermentation results differently [7], or these are interaction. The experiment using Central Composite Design (CCD) combined with neural network prediction model, is in order to obtain the optimum process conditions.
2. Materials and Methods
2.1. Materials
Seaweed industrial residues.
2.2. Major Equipments
Mill, the production from the Beijing Kun Jie Yu Cheng Machinery Co., Ltd., Model KCS-10; heated oven, the production from Kim Nam Instrument Factory, model DHG-9053A; Biochemical incubator, the production from Hai Qixin Scientific Instruments Co., Ltd., model LRH-70.
2.3. Main Experimental Methods
2.3.1. Fermentation Process
Seaweed residue was crushed through the mill to a particle diameter of 3 mm - 5 mm, then added cellulase, potassium dihydrogen phosphate, ammonium chloride, the EM inoculants (the EM effective microbial agents is a novel compound microbial preparation, brown translucent liquid, PH value between 3.5 to 4.5, the composite culture of photosynthetic bacteria, lactic acid bacteria, yeasts, actinomycetes bacteria, Acetobacter and other five families, 10 are over 80 kinds of anaerobic or suspected of oxygen normal microbiala), Indian meal, lactose and water, later mixed into the airtight container well-proportionally and placed in a biochemical incubator (25˚C) for fermentation, until fermentation is completed and then removed from the container, placed in heated oven (50˚C) to dry.
2.3.2. Determination of Protein and Polysaccharide Content
In this study, all the protein and polysaccharide content of samples are determined by Kjeldahl method (GB5511- 85) (nitrogen protein conversion factor of 5.95) and the phenol sulfuric acid method.
2.3.3. Experimental Design
The effect on fermentation products of cellulase, bacteria agents, ammonium chloride and number of days are investigated respectively. Combined with CCD and General Regression Neural Network (GRNN), protein content and polysaccharide content, two dependent variables, were analyzed to determine the extraction of the optimal process conditions.
2.4. Data Process Software
The experimental data was analyzed by the desingexpert6.5, and Matlab2009b software.
3. Results and Discussion
3.1. Effect of Basic Material on Fermentation
3.1.1. Effect of Temperature on Fermentation
Temperature is one of the most important factor to affect microbial growth and reproduction, same microorganisms has the different optimal temperature with variable physiological and biochemical processes, that means, the optimum growth temperature is not equal to the highest incubation temperature, neither does not mean fermentation speed is highest when incubation temperature nor the cumulative amount of metabolites when the incubation temperature, also does not mean the highest amount of incubation temperature either. For instance, Streptococcus thermophilus optimal growth temperature is of 37˚C, the optimal fermentation temperature is of 47˚C, the cumulative product of the optimum temperature is of 37˚C. From Figure 1, the protein content of the fermentation products gradually increased with increasing temperature at the range of 20˚C - 25˚C, reached the maximum at 25˚C, the protein content decreased with increasing temperature over 25˚C. Apparently microbial enzyme activity will be affected when temperature is higher than 25˚C, which leads to restraint of regular growth and reproduction, resulted in protein and crude fiber content of fermented products has been decreased with increasing temperature. Accordingly, the optimal temperature of the fermentation is of 25˚C.
3.1.2. Effect of Material-Water Ratio on Fermentation
Material-water ratios is an important influential factor in solid-state fermentation, extortionate water content will lead to aggregation of medium and cause lower porosity, affect the transfer of oxygen; while water content is too low, medium expansion will be retarded, which is not able to meet the needs of the microbial growth and metabolism, thereby inhibiting cell growth. The experiment has been carried out to study the effect of material-water ration on fermentation; results are shown in Figure 2. According to Figure 2, the material-water ratio had a greater influence on fermented products. Protein and polysaccharide increased gradually while material-water ratio from 50% - 65%, declined slightly at 70%, and de-
Figure 1. Effect of temperature on fermentation.
Figure 2. Effect of material-water ratio on fermentation.
clined sharply from 75% - 90%. Water-material ratio at the range of 50% - 65%, seaweed residue is of high hardness, bonded together in lumps, and turn to loose from 70% - 85% and semi-liquid at 90%, according to above analysis of data and phenomena, water-material ration at 65%, algae residues is of a fitting loose condition to maintain certain porosity and advantageous to interaction of water and air in solids, so that the dissolved oxygen can be delivered smoothly. Thus, the water-material ratio at 65%, the fermentation products is of the highest protein and polysaccharide content.
3.2. Single Factor Analysis
Basic raw materials: seaweed waste 1000 g, water 1500 g, potassium dihydrogen phosphate 10 g, Indian meal 15 g, lactose 15 g.
3.2.1. Effect of Cellulase on Fermentation
Under the condition of adding bacteria agents 10 g, ammonium chloride 15 g, fermentation of 10 days to study the effect of cellulase on fermentation. Cellulase can decompose raw materials in the natural cellulose into fermentable sugars, the other hand, cellulase can decompose the cell wall to release protein, starch and other nutrients in it, while the raw fiber can be degraded into the glucose which can be digested and absorbed by livestock. Figure 3 shows that protein and polysaccharide content of fermentation products is 9.58% and 47.25% respectively when added 6 g cellulase, while adding cellulase 10 g, protein and polysaccharide content reaches its maximum (12.68%, 58.45%), further addition of cellulase protein brought about a downward trend the content of protein and polysaccharide, this is due to the cellulose to cellulase adsorption sites has basically occupied, increasing the amount of enzyme can only increase the adsorption of enzyme invalid, and thus will not significantly im-
Figure 3. Effect of cellulase on fermentation.
prove the effect of fermentation; the other hand, enzyme fermentation environment of excessive make pH decreased, the agents normal metabolic activity, reduction of the fermentation product. Therefore, 10 g is an ideal addition amount of cellulase.
3.2.2. Effect of Bacterial Agents on Fermentation
Under the condition of adding cellulase 10 g, ammonium chloride 15 g, fermentation of 10 days to study the effect of bacterial agents on fermentation. Figure 4 shows that when less amount of agents was added, the effect of fermentation is poor, protein and polysaccharide content in fermentation products is only 9.23% and 44.85%, since the synergy and co-residence effect between the microbial, increasing amounts of agents will improve fermentation effect significantly, with the increasing amount of bacteria agents, the maximum protein and polysaccharide content (12.85%, 52.15%) was got by added agents 8 g, while added bacteria agents amounts further, protein and polysaccharide content presented a downward trend, this may occur because the autolysis of bacteria due to improvement of proteolytic enzyme activity or some bacteria produced antibiotics and other secondary metabolites. Therefore, the amount bacteria agent of about 10 g is an ideal.
3.2.3. Effect of Ammonium Chloride on Fermentation
Under the condition of adding cellulase 10 g, bacteria agent 8 g, fermentation of 10 days to study the effect of bacterial agents on fermentation. Adequate source of nitrogen are necessary for microbial propagation to synthesize such as amino acids, nucleic acids and other cellular material and to regulate metabolism, so necessary nitrogen should be added into fermentation, on the other hand, addition of inorganic nitrogen can promote microbial metabolism transformed it into protein, consequently improve the protein content in the algae residue directly.
Figure 4. Effect of bacterial agents on fermentation.
Figure 5 shows that, the best fermentation result was made when ammonium chloride dosage is of 15 g, the content of protein and polysaccharide was 14.85% and 54.25% respectively, while increasing ammonium chloride further did not affect the amount of the polysaccharide content, but will caused protein content to decrease significantly, this is because excess ammonium chloride make fermentation environment weakly acidic, thereby affecting the cell structure of bacteria, thus affecting isolates metabolism and product synthesis, so the amount of ammonium chloride, 15 g best.