Immobilized Cells in Food Industry

Winemaking and Brewing

In traditional winemaking, the natural microflora of the grapes, remaining only S. cerevisiae at the end, performs alcoholic fermentation of grape must. Nowadays specific, dry yeasts (mostly strains of S. cerevisiae) with different enzyme activities for the liberation of aroma compounds are added to start the fermentation. Winemaking is usually carried out in batch reactors, although continuous processes using immobilized cells resulted in higher productivity and high product yields. Packed bed reactors have the advantage to create anaerobic conditions ideal for ethanol fermentation. Support materials for cell immobilization in winemaking have to fulfil food safety considerations and must be non-toxic, food-grade materials. A lot of different techniques and materials have been used for yeast cell immobilization but considering the economic and food safety aspects, dried and sterilized plant materials remaining after juice pressing like grape stems and skins seem to be the best solution for adsorption of yeast cells. Consumers will also accept winemaking processes where only natural materials are used (Genisheva et al. 2014).

During maturation of wine, malolactic fermentation can take place where lactic acid bacteria convert L-malic acid to L-lactic acid by decreasing the acidity of the wine. Using immobilized Oenococcus oeni (formerly Leuconostoc oenos) doubled the degradation rate of malic acid, resulting in a milder taste in the wine (Genisheva et al. 2014).

In sparkling wines and champagnes, the removal of lees takes a lot of time. Using coated alginate beads for the immobilization of champagne yeasts makes it easy to separate the cells from the liquid (Genisheva et al. 2014).

Continuous beer fermentation was made by immobilizing brewer’s yeasts on cheap plant wastes like corncobs and spent grain, resulting in “quite acceptable” beer with unbalanced flavour compared to a traditional lager. By refining process parameters, the authors hoped to achieve better aroma and taste competing with traditional beer. Immobilization has shortened the fermentation time, reducing the costs of beer making (Branyik et al. 2005).

Dairy Products and Probiotics

Dairy products like yoghurts and cheeses are consumed in large quantities owing to their good taste and digestibility and health-promoting effects. Lactic acid bacteria (LAB) play a key role in the fermentation of milk to various products by coagulation of milk proteins through lactic acid production. Starters are mainly used in frozen form and are added directly to the milk. Problems with starters are arising from plasmid loss, sensitivity to bacteriophages, and contamination. Plasmid encoded products of LAB are involved in the degradation of the milk protein casein, production of lactic acid and flavour compounds. Plasmids are also responsible for production of bacteriocin and EPS, and for resistance to bacteriophages (Cui et al. 2015). Therefore, plasmid loss can cause starter failure. Immobilization of LAB improved plasmid stability, bacteriocin and EPS production, and resistance to bacteriophages (Doleyres and Lacroix 2004). Continuous production of single or mixed strains of LAB starters can be achieved by using cell immobilization techniques where the cells can leak from the support, ensuring high yield of living, biologically active cells (Doleyres and Lacroix 2004).

Probiotics are living microorganisms capable of colonizing the human gut and conferring physiological benefits to the host. Probiotics are mostly administrated by dairy products such as yoghurts. Not all LABs fulfil the requirements for probiotics; mainly Lactobacillus and Bifidobacterium species are used and added as probiotic starters. The suggested consumption of probiotics is 106-108 bacteria/g of food on a daily basis. Unfortunately, some probiotics, especially bifidobacteria, are very sensitive to the environmental changes during yoghurt fermentation, and the number of living bacteria decreases in the product. Bifidobacteria show also low survival rate during cold storage of dairy products; technologies ensuring stability of probiotics are therefore still a challenge for the food industry (Mitropoulou et al. 2013). Immobilization of probiotics can solve some problems by protecting cells from the pH and temperature changes, and increasing their viability and stability in fermented milk products, thus increasing their cell number. Another challenge for probiotics is the harsh environment in the human GI (gastrointestinal) tract. Low pH and protease activity in the gastric juice, followed by the emulsifying action of bile, cause cell death for the majority of microorganisms in the human GI tract. The number of surviving probiotics that are able to colonize the gut depends on their resistance to the above-mentioned circumstances. Immobilization can help the probiotics to survive. Different techniques and supports were used for the immobilization of LAB and probiotics with promising outcome. Delignified wheat bran was used for the adsorption of Lactobacillus casei as probiotic and L. bulgaricus as starter. The strains were immobilized separately and freeze-dried after the process. The freeze-dried, immobilized strains were used for yoghurt fermentation. Not only the cell viability was increased during storage at 4°C but also the aroma was more complex and preferred by tasters compared to traditionally produced yoghurt. The immobilized, frozen cells showed also higher survival in simulated gastric juice compared to non-immobilized ones (Terpou et al. 2017).

Vinegar Production

One of the oldest processes using the application of immobilized cells is vinegar production. Although chemical synthesis of acetic acid is cheap and can provide large quantities, only acetic acid fermented by microorganisms is allowed for human consumption. Acetic acid bacteria oxidize ethanol to acetic acid in a two-step process. The source of ethanol is mostly wine and the product is called vinegar. The traditional Orleans Process was used since 1670 in France where wooden casks were filled with wine and vinegar was added to start the process. The acetic acid bacteria flocculated and formed a thick slime on the walls of the barrel. In 1832 the Generator Process was developed where the bacteria were immobilized mainly on beech wood shavings. Fermentation was performed in open, large volume tanks. Although nowadays submerged cultures are used for vinegar production, the traditional method with immobilized cells is still used for manufacturing fine products (Bhat et al. 2014).

Enzyme Production with Immobilized Cells

In the modern food industry, enzymes are used in many fields to facilitate processes or to improve product quality. Microorganisms are cheap sources of enzymes because they can be cultured at industrial scale using agricultural waste materials as substrate. Lipases can be used in bidirectional actions, i.e. for hydrolysis or synthesis of lipids. Aspergillus niger is one of the best-known lipase producers. The spores of the mould were immobilized in sodium alginate beads and wheat bran was used as substrate. The alginate beads were reused in four subsequent fermentation cycles, giving higher yield than free cells. Because lipase is an intracellular enzyme, the application of immobilized whole cells can eliminate the steps of enzyme extraction and purification, making the enzymatic process more economical (Chandorkar et al. 2014).

Immobilized Enzymes in the Food Industry

As mentioned before, enzymes are used in many processes in the food industry'. Since the purification of individual enzymes is a time-consuming multi-step process with high costs, sometimes crude extracts or whole, non-viable cells are used. Reusable enzymes can reduce the costs and make these processes economical. Immobilization of the enzymes offers the possibility of recovering the biocatalyst if that is needed.

In some cases, the added enzyme becomes a part of the product, e.g. proteases or amylases used in improving the quality of wheat flour will remain in the baked product without the chance of reuse.

Starch Industry and Production of Sweeteners

Starch processing requires a lot of enzymes for the hydrolysis of starch and refining the products. Alfa amylases create malto-dextrins by partial hydrolysis of starch; thereof in the saccharification process glucoamylases create glucose syrup. Pullulanase debranches amylopectin by the hydrolysis of al-6 bonds. Immobilization of a-amylases, glucoamylases or pullulanase resulted in better thermal stability of the enzymes but Km values usually increased, showing that conformational changes during immobilization, especially by cross-linking or covalent binding of the enzymes, decreased enzyme activity (Contesini et al. 2013).

Isomaltulose (6-O-a-D-glucopyronosy 1-1 -6-D-fructofuranose) is a sweetener with low cariogenicity and glycemic index, made from sucrose by the enzyme a-glucosy ltransferase. The enzyme is produced by several bacterial strains. Glucosyltransferase from Erwinia sp. was used for immobilization in Ca-alginate beads in the form of crude extract or whole cells lysed by ultrasonication (Kawaguti and Sato 2011). Lysed cells showed higher stability than the crude enzyme extract, and reached 53%-59% conversion of sucrose to isomaltulose.

In the production of the sweetener aspartame, aspartic acid and phenylalanine- methyl ester are linked together. E. coli entrapped in к-carrageenan gel produces aspartic acid. In a few days after immobilization. E. coli die and the aspartase enzyme releases from the disintegrated cells and can be used for years as immobilized enzyme (Johnson-Green 2002).

The by-product of cheese fermentation is whey with high lactose content. (3-galactosidase convert lactose into glucose and galactose, yielding a product sweeter than lactose. With the aid of (3-galactosidase, whey can be converted to a sweet syrup usable in the food industry. Also, lactose in milk can be hydrolysed by the enzyme, resulting in lactose-free milk which has two advantages: people with lactose intolerance can drink this milk, and it can be used for production of ice-cream, condensed milk, etc., where lactose crystals would give a gritty texture. The enzyme is produced by several microorganisms. Enzyme from yeasts like Kluyveromyces lactis are used for the hydrolysis of milk lactose, while fungal (3-galactosidases are used for the hydrolysis of acidic whey. The enzyme was immobilized by covalent binding or entrapment in к-carrageenan or polyvinyl alcohol gels. The results are promising but there is a problem of microbial contamination in continuous systems. To avoid intermittent sanitation of the equipment, the industry shows interest in thermostable or cold-active (3-galactosidases (Grosova et al. 2008).

Production of Prebiotics

Prebiotics are non-digestible food components that help maintain the healthy gut microflora. Galacto-oligosaccharides (GOS) contain usually 1-5 molecules of galactose and one glucose. GOS is synthetized by the reverse reaction of (3-galactosidase, named transgalactosylation. This reaction needs high lactose concentration, high temperature, and lack of water. These circumstances are better achieved in immobilized systems where the enzyme is protected from heat and substrate inhibition. Maximum GOS production (55% w/w) was reached by p-galactoside, entrapped in chitosan beads, in a packed bed reactor (Grosova et al. 2008). Using magnetic polysiloxane- polyvinyl alcohol for immobilization of the enzyme, the biocatalyst was collected from the fermentation broth by magnetic field and was reused 10 times with only minimal loss of activity (Contesini et al. 2013).

Immobilized Enzymes in the Production of Beverages

Glucosidases are used for aroma liberation in wine because most of the volatile aroma compounds are linked to sugars by glycosidic bounds. P-glucosidase from Aspergillus sp. was immobilized in a sol-gel support, resulting in improved thermal and pH stability but also increased Km due to mass transfer limitations. To improve the productivity of juice pressing, immobilized pectin lyase from Penicillium italicum was immobilized by covalent binding to Nylon 6. Immobilization increased the storage and thermal stability of the enzyme (Contesini et al. 2013). Naringinase is used for debittering of citrus fruit juices. Fungal naringinase was immobilized on different supports like polyvinyl alcohol cryogels, cellulose triacetate nanofibres etc. Acetolactate in beer protracts the maturation to 2-12 weeks. Using free or immobilized a-acetolactate decarboxylase, maturation can be shortened to 1 day (Raveendran et al. 2018).

Conclusion

Immobilization of cells or enzymes in the food industry is mainly in the developing phase, although some applications are used in industrial scale (Table 8.1). The most promising area is the microencapsulation of probiotics that offers protection against

TABLE 8.1

Recent Examples on the Application of Immobilized Cells and Enzymes in the Production of Food Industrial Products, Beverages and Starter Cultures

Product

Microorganism or Enzyme

Immobilization Method

References

Wine

S. cerevisiae Oenococcus oeni

Adsorption on plant wastes Entrapment in Ca-alginate beads

Genisheva et al. (2014)

Champagne

S. cerevisiae

Entrapment in coated Ca-alginate beads

Genisheva et al. (2014)

Beer

S. cerevisiae

Adsorption on plant wastes

Branyik et al. (2005)

Starter cultures

Lactic acid bacteria

Entrapment in food-grade polymer beads

Doleyres and Lacroix (2004)

Probiotics

L. casei Bifidobacteria

Adsorption on delignified wheat bran

Terpou et al. (2017)

Lipase

A. niger

Entrapment in Ca-alginate beads

Chandorkar et al. (2014)

Isomaltulose

Erwinia sp.

Entrapment in Ca-alginate beads

Kawaguti and Sato (2011)

Galacto-oligosaccharides

p-galactosidase

Entrapment in chitosan beads

Grosova et al. (2008)

the cell limiting factors arising during dairy production or in the human GI tract. Immobilization of enzymes or living or dead cells containing these enzymes make the processes cheaper and more feasible. In some cases, advantages of immobilization are contrasted by the mass transfer limitations caused by the support material; and choosing the suitable material is also a difficult issue, especially from a food safety point of view. Immobilization needs redesigning the process flow and equipment used but offers an economical production by reusing the biocatalyst. Taking pros and cons into account, we can say that immobilization will find its way in the food industry.

ACKNOWLEDGEMENT

MT thanks to the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences.

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