Enzymatic processes and enzymatic extraction

Glucose

Glucose is the most 'popular' conversion product from food wastes, since glucose is the monomer for cellulose and starch. Cellulose degradation is usually carried out by complex cellulolytic enzyme preparations (like Celluclast, or Rapidase, etc.), containing several types of cellulases. Trichoderma reesei (earlier Trichoderma viridae) are considered to be the best sources of cellulase enzymes. However, the mixture should often be supplemented with P-glucosidase enzyme to enhance the hydrolytic process. Any kind of cellulose-containing waste or co-product is considered for glucose production, e.g. rice, wheat and rye straw, corn cob (Vlasenko et al., 1997; Kaur et al, 1998; Hang and Woodams, 2001; Sun and Cheng, 2005). These substrates need pre-treatment (acidic or steam treatments) in most cases.

Starch is much easier to degrade than cellulose, thus the majority of the glucose comes from various starch-containing waste sources, as produced in potato, cassava and corn manufacturing (Jin et al., 1998; Gao et al., 2002; Del Re et al., 2003). Amylases: mainly alpha- and beta-amylases (sometimes glucoamylases) from various sources (often Aspergillus species) are used for enzymatic starch hydrolysis. Recently, selection of thermostable amylase enzymes has been the focus of the research in this field. According to the cost analysis of these processes, enzymes are the most expensive materials. To reduce the cost, 'on-site' enzyme production is worthy of further study and development, as mentioned above.

A special bioreactor system is indicated from a practical point of view, due to the strong inhibition phenomena occurring in enzymatic polysac-charide hydrolysis. One of the most promising solutions to the problem is the utilisation of membrane bioreactors (Belafi-Bako et al., 2002a, 2006). In these bioreactors the enzymatic reaction and the separation of the inhibitory product (glucose) takes place simultaneously in one unit. The differences in size of the substrate (polysaccharide) and the product make separation possible by a suitable porous membrane (ultrafiltration range); this is able to reject long polysaccharide chains as well as the biocatalyst, while the product passes through the membrane easily. In such a system, continuous uptake of substrate and release of product without loss of enzyme can be achieved. Moreover, higher effectiveness can be obtained in this particular system because of the lack of product inhibition.

Ethanol

Ethanol is one of the most important glucose-based products and is either available as an ingredient of wastes and co-products in the food industry (such as in molasses from the sugar industry) or can be obtained by hydrolysis from wastes (such as corn stover) containing polysaccharides. Several techniques have been developed according to the raw material used. One of the simplest examples is the direct fermentation of molasses by Saccharomyces cerevisiae. If polysaccharides are the raw materials, sac-charification and fermentation can be applied as separate steps or simultaneously (Varga et al., 2004).

Protein hydrolysates

Protein hydrolysates (mixtures of amino acids and oligopeptides) can be produced by protease enzymes. A detailed method for the manufacture of gelatine and gelatine hydrolysates is described by Birch et al. (1981), using collagen-rich wastes from abattoirs. An Alcalase enzyme preparation is used at a temperature of 28 °C and the conditioning time is 6-24 h. Gelatine hydrolysates are produced by hydrolysis using Alcalase and Neutralase preparations. Recently, keratinases from various sources have been the focus of many investigations. Certain keratinases (for example from Paecilomyces marquandii, Doratomyces microsporus, or Chryseobacterium sp.) are able to degrade feather with an acceptable reaction rate (Brandelli and Riff el, 2005; Gradisar et al., 2005); this is regarded as an extremely high-impact finding due to the huge amount of feather produced as waste worldwide. Although feather in its original form is considered as a low biological value protein source - due to its deficiencies in nutritionally essential amino acids like methionine, lysine, histidine and tryptophan - the feather meal obtained by thermal processing has been incorporated into the diets of certain animals (Onifade et al., 1998). Microbiological degradation of feathers, however, may enhance the nutritional value of feather protein hydrolysate, since the biomass could autolytically contribute to the protein and amino acids content of the feather meal. Additionally, certain keratinophilic bacteria (e.g. Kocuria rosea) also synthesise carotenoids, which are useful compounds in salmonid feed or egg yolk pigments (Bertsch and Coello, 2005).

Proteases are also used for bioprocessing of another enormous waste mass, i.e. crustacean (shrimp, crab, lobster, etc.) shells. Deproteinisation of shrimp and crab shells has been carried out using the Alcalase enzyme preparation and resulted in a valuable chitin source (Oh et al., 2000). Recently, an enhanced method was presented where protein hydrolysate can be recovered as well (Synowiecki and Al-Khateb, 2000; Gildberg and Stenberg, 2001); thus the effectiveness of the process is improved.

Other types of animal wastes may be similarly good sources of protein hydrolysates. Atlantic cod viscera (Aspmo et al., 2005), chicken intestinal waste (Jamdar and Harikumar, 2005), shrimp heads (Ruttanapornvapee-sakul, 2005), hake filleting waste (Martone et al., 2005) and ram horn (Kurbanoglu and Algur, 2004) are just a few examples of the wastes available from animal processing. The protein content of these materials can be hydrolysed by various commercial protease enzyme preparations (such as Alcalase, Neutrase, or Papain) or by different endo- and exopeptidases from Aspergillus and Bacillus species.

Utilisation of these protein hydrolysates (Table 9.4) can be divided into two main areas according to whether the nutritional or the biodegradable properties are exploited. Protein hydrolysates (concentrates) can be used either as nutrient sources in media for microorganisms and feed supplements for higher animals, or as components for polymer modifications (improving biodegradability).

Natural flavour compounds

Natural flavour compounds can be synthesised from a co-product formed in distilleries (alcohol production). It is called fusel oil, and contains ethanol and short chain alcohols. Volatile, flavour ('fruit') esters can be manufactured if these alcohol compounds are reacted with short chain acids,

Table 9.4 Utilisation of protein hydrolysates

Sources

Ram horn

Hake (Merluccius hubssi)

filleting waste Shrimp (Pandalus borealis) wastes Shrimp (P. semisulcatus)

head wastes Sardinella aurita fish wastes Leather waste of tanning

Leather waste (chrome shaving) Shrimp head silage

Fish wastes and Feather

Utilisation

Medium for aerobic bacteria

Reference

Kurbanoglu and

Algur, 2004 Martone et al., 2005

Gildberg and

Sternberg, 2001 Mizani et al., 2005

Ghorbel, 2005

Saha et al., 2003

Kresalkova et al., 2002

Plascencia-Jatomea et al., 2002 Langar et al., 1993

Nutrient source in media for bacteria and archae Feed supplement for salmonid fishes

Animal/aquaculture feed formulations Nitrogen source for Rhizopus oryzae (lipase production) Modification of linear-low-density polyethylene polymer Modification of polyvinyl alcohol films Protein source for Nile tilapia

(Oreaochromis niloticus) Protein sources for sea bass fry (Dicentrarchus labrax)

resulting in low molecular weight esters. The reaction is catalysed by lipase enzyme and can be carried out in organic solvent or in a solvent-free system (Gubicza et al., 2000; Ehrenstein et al., 2003).

Biolubricants

Biolubricants can also be manufactured from fusel oil. In this case, longer chain acid compounds (fatty acids from, for example, hydrolysis of plant oils) should be used in the esterification reaction. The process can be carried out by acidic (Ozgulsun et al., 2000) or enzymatic catalysis (Fig. 9.1). The drawback of acidic catalysis is that acid traces may remain in the

Types of biomass f. f

Wood A Crops

Landfill gas Alcohol fuels

Glycerol c

Oil hydrolysis

Biolubricant (acid traces)

Acid-catalysed esterification s/

Biolubricant (acid traces)

Landfill gas Alcohol fuels

Oil hydrolysis

Acid-catalysed esterification s/

Ethanol production

Enzyme-catalysed esterification s/

Ethanol production

Enzyme-catalysed esterification s/

Biolubricant (acid-free)

Ethanol

Biolubricant (acid-free)

Fig. 9.1 Production of biolubricants.

product, which causes corrosion during utilisation. By applying enzymatic catalysis (e.g. lipase), this can be avoided. The biolubricants obtained are not only derived from natural renewable sources (biomass), but can also be degraded biologically (Dormo et al., 2004).

Similar biolubricants can be produced by enzymatic extraction of ethanol (from food wastes) by fatty acids (Csanyi et al., 2004). Ethanol recovery from the aqueous fermentation broth can be realised by extraction using, for example, oleic acid, with simultaneous esterification by lipase enzyme. In this way, ethyl oleate is manufactured, while ethanol inhibition is avoided.

According to tribological tests, these (ester) types of biolubricants can be characterised as lubricants having a low flash point, low pour point, high viscosity index, and low acid number which is a result of the enzymatic catalysis (instead of acidic catalysis). Thus, it is suggested that they are used mainly at high-speed, and low-load tribological regimes. In industry they can be applied as, for example, cooling lubricant compounds for metal-working processes, and also in particular processes where lubricant loss may occur, e.g. mist lubrication, chain lubrication and launch engine lubrication.

Galacturonic acid

Galacturonic acid is an acidifying agent in foods and the monomer of pectin molecules. Thus pectin-containing co-product and waste can be processed to recover the pectin. Sugar beet pulp is one of the raw materials most often used (Jordening et al., 2002). When pectin is extracted from sugar beet pulp by hot water, the process can be promoted by using pectinase enzymes. In this way pectin becomes more soluble, its viscosity is decreased and the yield of the enzymatic extraction is much higher. If the aim is recovery of galacturonic acid, complete degradation of pectin can be carried out. However, controlling the enzyme action may result in partial degradation, where soluble pectin can be obtained.

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