SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Viability of probiotic bacteria during the production and storage of fermented milks is the most important topic of discussion in the dairy industry. Addition of microalgae into milk for the production of fermented milk in order to enhance the viability of probiotics has been the subject of recent research. Spirulina and Chlorella are the most widely noted microalgae for fermented milks. They affect not only the viability of probiotics in final product but also the sensory attributes of them. Incorporation of microalgae into probiotic fermented milks along with enhancing the viability of probiotics would increase their functional characteristic. This is because they contain a wide range of nutrients and nutraceuticals and are considered as “functional food.” This article reviews the effects of supplementation of Spirulina platensis and Chlorella vulgaris into probiotic fermented milks on their different quality characteristics.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Probiotics are live microorganisms (bacteria or yeasts) which provide their host with health benefits. They do it by maintaining and/or improving the microbial balance of the intestinal medium if administered in adequate amounts (Fuller 1989; Gismondo and others 1999; Holzapfel and Schillinger 2001; Shah 2001). There are many reports describing probiotic health-promoting effects on gastrointestinal infections, antimicrobial and antiviral activity, improvement in lactose metabolism, reduction in serum cholesterol level and blood pressure, improvement in mineral absorption, stabilization of the gut mucosal barrier, antimutagenic and anticarcinogenic properties, immune system stimulation, antidiarrheal and anticonstipation properties, urogenital infections, atopic diseases, improvement in inflammatory bowel disease, and suppression of Helicobacter pylori infection by addition of selected strains to food products (Nakasawa and Hosono 1992; Wood 1992; Nighswonger 1996; Kailasapathy and Rybka 1997; Marth 1998; Riordan and Fitzgerald 1998; Salmine and Wrigth 1998; Sanders 1999; Saarela and others 2000). Certain species of lactobacilli and bifidobacteria are by far the most important probiotics used in probiotic products and currently, many types of these products (in particular probiotic dairy products) are available in most world markets (Hoier 1992; Mortazavian and others 2007a).

To obtain the claimed probiotic benefits, these bacteria must be viable and abundant at the time of consumption, and this is called “viability” (Gomes and others 1995). Although no worldwide agreement has been reached on recommended levels, generally the values of 106 CFU/mL and 107 or 108 CFU/mL have been accepted as the minimum and satisfactory levels, respectively (Karimi and others 2010; Sohrabvandi and others 2010; Mohammadi and others 2011). However, a major factor in the production of probiotic fermented milks is loss of viability of probiotics during the fermentation process, as well as during the refrigerated storage (Mortazavian and others 2005, 2006, 2007b, 2008, 2010, 2011; Nobakhti and others 2008; Shafiee and others 2010; Ahmadi and others 2001; Heydari and others 2011; Sadaghdar and others 2012) and these organisms often show poor viability when marketed (Klaver and others 1993; Dave and shah 1997; Ravula and shah 1998; Mortazavian and others 2007c). Numerous factors influencing the viability of probiotic cultures in fermented milks are as follows: pH, titratable acidity, the presence of other microorganisms, temperature, oxygen content, nutrients and growth factors, food additives, application of new technologies such as microencapsulation, and formulation of products (Shah 2000; Gueimonde and others 2004; Mortazavian and others 2007d, 2009; Cruz and others 2010; Mohammadi and Mortazavian 2010; Korbekandi and others 2011 ).

Recently, a trend has been started to add microalgae (cyanobacterial biomasses) into fermented milks in order to increase the functional product characteristics via promoting viability of probiotics as well as to enhance the nutritional attributes (Varga and Szigeti 1998; Varga and others 2002). Spirulina and Chlorella are blue-green microalgae which contain high-antioxidant components, abundant amino acids, high-quality proteins, Fe and Ca, unsaturated fatty acids, and many types of vitamins including A, B2, B6, B8, B12, E, and K. They have antiviral, anti-inflammatory, and antitumoral effects and reduce blood lipid profile, blood sugar, body weight, and wound healing time. Therefore, they are known as therapeutic and functional food (Fox 1986; Dillon and Phan 1993; Parada and others 1998; Kreitlow and others 1999; De Caire and others 2000; Merchant and Andre 2001; Gyenis and others 2005). It seems that the co-addition of microalgae and probiotics stimulates growth and increases viability and acid production of the probiotic bacteria (Shirota and others 1964; Stengel 1970; Zielke and others 1978; Kurita and others 1979; Webb 1982). On the other hand, microalgae present in fermented milks will affect the sensory properties of the final product. This article reviews the effects of supplementation of Spirulina platensis and Chlorella vulgaris (currently the most common applied microalgae) into probiotic fermented milks on their various quality characteristics.

Fermented Milks as Probiotic Food Carrier

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Functional foods are foods that have at least a certain nutritional highlight in addition to their regular nutritional properties along with having confirmed medicinal outcome to the consumer. They are produced by adding at least a chemical or microbial ingredient to a food-base, such as milk and milk products. Probiotics and synbiotics are functional foods with microbial enrichment (Mohammadi and Mortazavian 2011; Sedef Nehir and Simsek 2012).

One of the best food-bases for production of functional foods is fermented milks, due to the fact that they are inherently known as healthy foods, are regularly consumed by the vast majority of people in their long-term diet, possess pleasant sensory acceptance, and have extended shelf life which facilitates their distribution, sale, and consumption. Fermented milks are widely manufactured throughout the world. Approximately 400 generic names are applied to the traditional and commercial products; but in actual essence the list may include only a few variations. Yogurt is considered as the most popular fermented milk in the world (Korbekandi and others 2011).

The global market for probiotic ingredients, supplements, and foods was worth $14.9 billion in 2007 and reached USD16 billion in 2008. Estimates target a total of USD19.6 billion on sales in 2013. Food applications for probiotics are found in abundance in dairy products, with yogurts, kefir, and cultured drinks representing the major categories. Yogurt products accounted for the largest share of sales, represent 36.6%, and scientific development of which has shown the high sensory acceptance emerging food applications include probiotic cheese and ice creams, nutrition bars, breakfast cereal, infant formula, and many others (Cruz and others 2009a, 2009b; Granato and others 2010a, 2010b).

“Probiotic fermented milks” are considered as the most popular and investigated probiotic functional foods. Moreover, they are the most-consumed probiotic products. The critical value in these products is maintaining the viability of probiotics over the standard limits according to the national legislations until the end of shelf life. However, the presence of detrimental environmental factors in above-mentioned products (for example, low pH values, relatively high titratable acidity and redox potential, molecular oxygen, antimicrobial agents such as hydrogen peroxide, bacteriocins, short-chain fatty acids, some flavoring agents, salt, sugar, sweeteners and additives, and microbial competitions) usually causes the loss of viability in probiotics during fermentation and over the storage period. In addition, even the milk media (although is much more suitable than other types of nondairy fermented products) suffers from the lack of some nutrients for probiotics in an available form (Cruz and others 2010; Mohammadi and Mortazavian 2011; 2012a; 2012b). Therefore, many investigations in the field of probiotic technology have focused on the fortification of basic food media by means of growth promoting factors for mentioned microorganisms.

In addition to the viability of probiotics in cheese, sensory characteristics (flavor, texture, and appearance) have their special position and importance in total acceptance from consumers’ point of view. Studies have shown that flavor is the 1st indicator with respect to the choice of a food, followed by health considerations. These studies likewise indicated that consumers are not interested in consuming a functional food if the added ingredients confer disagreeable flavors on the product, even if this results in advantages with respect to their health (Cruz and others 2010; Granato and others 2010b). Therefore, the effects of mentioned factors on sensory profile of probiotic fermented milks must be evaluated in parallel with viability investigations after the addition of probiotic strains to fermented milks as well as incorporating the chemical ingredients for promoting their survival.

The Spirulina and Chlorella Usage and Their Functional Ingredients

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Cyanobacteria or blue-green algae are photoautotrophic microorganisms widely distributed in nature (Parada and others 1998; Molnár and others 2005). They are microscopic plants more closely akin to bacteria than to seaweed. Microalgae as photosynthetic microorganisms are considered as a very diverse group of organisms consisting of both prokaryotic and eukaryotic forms (Kreitlow and others 1999; Molnár and others 2009). They are an excellent source of high-value compounds and systematic screening for therapeutic substances, particularly from cyanobacteria, has received great attention (Barsanti and Goaltieri 2006). The current use of these resources has 3 aspects: tradition, scientific and technological development, and the so-called “green tendency” (Henrikson 1994).

From the scientific point of view, the cultivation of microalgae like Spirulina maxima began in 1919 with Warburg's investigations (Richmind 1992). In 1950, the United States and Japan began the experimental cultivation of this microorganism to investigate its chemical composition and possible industrial applications. From 1970 on, nutritional and medicinal studies on Spirulina have proliferated (Saxena and others 1983; Schwartz and Shklar 1987; Fox 1993; Chamorro and others 1996; Hayashi 1996). In 1970, massive annual production of microalgae, which could be used in protein production and in water treatment, was reported (Ciferri and Tiboni 1985; Ayala and Vargas 1987; Lacaz and Nascimento 1990; Canizares and others 1993; Oxa and Rios 1998). Spray-dried microalgal biomass typically contains 3% to 7% moisture, 46% to 63% protein, 8% to 17% carbohydrates, 4% to 22% lipids, 2% to 4% nucleic acid, 7% to 10% ash, and a wide range of vitamins and other biologically active substances (Gyenis and others 2005; Molnár and others 2009).

Therapeutic effects and nutrients of microalgae are shown in Figure 1. Below, the main characteristics and functional ingredients of S. platensis and C. vulgaris are discussed.

image

Figure 1. Therapeutic effects and nutrients of microalgae.

Download figure to PowerPoint

Spirulina platensis

Spirulina spp. (2 main species being S. platensis and S. maxima) are multicellular, filamentous cyanobacteria which have the appearance of coils in cross section. Spirulina spp. have been used for over 1000 y as a food source, some of which have a protein content as high as 55% to 70% of the total dry weight (Fuller 1989; Shah 2001). S. platensis is the dried biomass of Arthrospira platensis, which is a planktonic cyanobacterium that forms massive populations in tropical and subtropical water bodies characterized by high levels of carbonate and bicarbonate and high pH (up to 11) (Gyenis and others 2005; Molnár and others 2009). It contains 18 of the 20 known amino acids, high-quality proteins, more calcium than milk, more vitamin B12 than cow liver, vitamins A, B2, B6, E, H, and K, and all essential minerals, trace elements, as well as enzymes (Fox 1986).

It has also been shown that Spirulina is an excellent source of proteins (60% to 70% of its dry weight), vitamins, and minerals (Ciferri 1983; Parada and others 1998; De Caire and others 2000; Shimamatsu 2004; Spolaore and others 2006). The nutritive value of a protein is related to the quality of its amino acids, digestibility coefficient, as well as its biological value (Richmond 1984; Dillon and Phan 1993). Spirulina contains essential amino acids; the highest values of which are leucine (10.9% of total amino acids), valine (7.5%), and isoleucine (6.8%) (Cohen 1997).

Spirulina has a relatively higher provitamin A concentration (Belay 1997). An excessive dose of β-carotene may be toxic, but when the β-carotene is ingested from Spirulina or another vegetable it is usually harmless since the human organism converts it into vitamin A, up to the quantity it needs (Henrikson 1994). Spirulina is a very rich source of vitamin B12, and that is why these cyanobacteria are of great value for people needing supplements in the treatment of pernicious anemia (Becker 1984; Richmond 1984; Belay 1997).

Spirulina contains 4% to 7% lipids and essential fatty acids such as linoleic acid (LA) and γ-linolenic acid (GLA) (Othes and Pire 2001). The latter is claimed to have medicinal properties and prerequisite for arachidonic acid and prostaglandin synthesis (Dubacq and Pham-quoc 1993). GLA lowers low-density lipoprotein, and is 170-fold more effective than LA (Cohen 1997; Sánchez and others 2003). The fatty acid composition of Spirulina is generally characterized by high levels of the ω-6 series (De Caire and others 2000). Deficiency in linolenic acid is associated with vision (Neuringer and Connor 1986) and nervous system defects, regulation of blood pressure, cholesterol synthesis, inflammation, and cell proliferation (De Caire and others 2000; Gyenis and others 2005).

Iron is not appropriately absorbed in some nutritional complements. Iron is 60% better absorbed in Spirulina than ferrous sulfate and other components. Consequently, it could represent an adequate source of iron for anemic pregnant women (De Caire and others 2000).

S. platensis contains about 13.6% carbohydrates; some of which are glucose, rhamnose, mannose, xylose, and galactose (Shekharam and others 1987). Spirulina does not have cellulose in its cell wall, a feature that makes it an appropriate and important foodstuff for patirnts who have poor intestinal absorption and for geriatric patients (Richmond 1984). A new high-molecular-weight polysaccharide with immunostimulatory activity has been isolated from Spirulina and is called “Immulina.” This highly water-soluble polysaccharide represents the dry matter between 0.5% and 2.0% (w/w) (Pugh and others 2001).

 Some natural pigments are found in Spirulina, which are responsible for the characteristic colors of certain flamingo species that consume these cyanobacteria in the African Valley. This knowledge has promoted the use of this microorganism as a source of pigmentation for fish, eggs, and chicken (Ciferri 1983; Saxena and others 1983; Henrikson 1994). Spirulina also increases the yellowness and redness of broiled chicken due to accumulation of the pigment (Toyomizu and others 2001; Sánchez and others 2003). Spirulina (including Spirulina maxima and S. platensis) contains phycocyanin. Phycocyanin is a nontoxic blue pigment in Spirulina and can act as a free radical scavenger and a powerful antioxidant. Studies have shown that phycocyanin can exert a wide range of anti-inflammatory effects. It has been claimed that consumption of Spirulina is beneficial to health due to its chemical composition (Fox 1986; Doumenge 1993; Henrikson 1994; Parada and others 1998).

There are data showing that Spirulina has various possible health-promoting effects: the alleviation of hyperlipidemia, suppression of hypertension, protection against renal failure, growth promotion of intestinal Lactobacillus, and suppression of elevated serum glucose level (De Caire and others 2000; Shimamatsu 2004; Spolaore and others 2006). Spirulina possesses some antiviral and antitumor properties. One of the main concerns about the consumption of microorganisms is their high content of nucleic acids that may cause diseases such as gout. Spirulina contains 2.2% to 3.5% RNA and 0.6% to 1% DNA, which represents less than 5% of these acids, based on dry weight. These values are smaller than those of other microalgae like Chlorella and Scenedesmus (Ciferri 1983).

Chlorella vulgaris

Chlorella is a genus of single-celled (unicellular) green algae belonging to the phylum Chlorophyta. It is spherical in shape, about 2 to 10 μm in diameter, and without flagella. Chlorella contains the green photosynthetic pigments chlorophyll-a and -b in its chloroplast. It multiplies through photosynthesis and requires only carbon dioxide, water, sunlight, and a small amount of minerals to reproduce (Scheffler 2007).

Chlorella is a good source of nutrients such as valuable protein, calories, fat, and vitamins (Belasco 1997). Under certain growing conditions, Chlorella yields oils high in polyunsaturated fats; for example, Chlorella minutissima yielded EPA at 39.9% of total lipids. It also produces astaxanthin, canthaxanthin, and, in minor amounts, β-carotene (Mendes and others 2003; Gyenis and others 2005). C. vulgaris contains other antioxidants such as lutein and chlorophyle. It is a rich nutritional ingredient because it contains 61.6% proteins, 12.5% fat, 13.7% carbohydrates, trace elements (aluminum, selenium, phosphorus, zinc, calcium, and magnesium), and vitamins (thiamine, B1, B2, B6, ascorbic acid, D, E, and K) (Valdivia and others 2011).

Chlorella is a good source of polyunsaturated fatty acids (PUFAs) (38.94%) and has 13.32% of total lipids. Palmitic acid content of Chlorella was 15.41%, EPA (20:5n-3) content, 3.23%, and docosapentaenoic acid, 3.11%. Docosahexaenoic acid (C22:6n-3) content of Chlorella was extremely high (20.94%). PUFA content of Chlorella was 38.94% and the total omega-3 (n-3) level was 29.21%. Chlorella was rich in P (1761.5 mg), Na (1346.4 mg), K (749.9 mg), Ca (593.7 mg), mg (344.3 mg), and Fe (259.1 mg). Other mineral contents included Mn (2.09 mg), Zn (1.19 mg), Se (0.07 mg), Cu (0.06 mg), and Cr (0.02 mg) (Oglu and Ünal 2003).

It has been demonstrated that Chlorella exhibits various immunological effects such as antibacterial and antiviral action. Furthermore, administration of Chlorella prevents cadmium-induced toxicity and toxin-caused oxidative stress and cellular damage. C. vulgaris administration maintains the renal cytoarchitecture against HgCl2-caused oxidative stress and nephrotoxicity (Valdivia and others 2011).

Numerous references on the health benefits of consuming Chlorella spp. have shown that their ingested extracts can lower blood sugar levels, increase hemoglobin concentrations, act as hypocholesterolemic and hepatoprotective agents during malnutrition, and reduce ethionine intoxication (Schwartz and others 1990; Yamaguchi 1996; Watanabe and others 2002; Barrow and Shahidi 2008). Intake of Chlorella was beneficial to the improvement of insulin sensitivity in normal Wistar rats (Jeong and others 2009).

Feeding microalgae to elderly people or animals has been demonstrated to protect against age-dependent diseases, particularly cardiac hypertension or hiperlipidemia. C. vulgaris has also been proved to have immune-modulating and anticancer properties (Janczyk and others 2007).

Chlorella has been found to have antitumor properties when fed to mice (Janczyk and others 2007; Jeong and others 2009). Another study found enhanced vascular function in hypertensive rats given oral doses of Chlorella (Belasco 1997). Clinical studies on Chlorella suggest effects including dioxin detoxification in humans (Nakano and others 2005) and animals (Takekoshi and others 2005), healing from radiation exposure in animals (Singh and others 1995), and the ability to reduce high blood pressure, lower serum cholesterol levels, accelerate wound healing, and enhance immune functions in humans (Merchant and Andre 2001). Among eukaryotic microalgae, a glycoprotein prepared from C. vulgaris culture supernatant exhibited protective activity against tumor metastasis and chemotherapy-induced immunosuppression in mice (Barsanti and Goaltieri 2006).

Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Several studies have indicated that the chemical characteristics (pH, acid production) of fermented products such as yogurt and probiotic fermented milk products improved due to supplementation with prebiotics such as inulin, resistant starch, fiber and calcium, date fiber, β-glucan, glucose, and raffinose. This could be due to the nutritional benefits of prebiotics in enhancing the growth of probiotics and promoting acid production during fermentation and storage (Zare and Orsat 2012).

In a study by Molnár and others (2009), changes in acid production of mesophilic lactic acid bacteria grown in milk were investigated. Milk samples enriched with Spirulina at different concentrations (0%, 0.3%, 0.5%, or 0.8%) were inoculated at the rate of 1% with the mesophilic LAB strains to be tested. The pH value was measured at regular intervals (every 2 h) with pH 4.01 and 7.01 standard buffer solutions. Results of this study showed that Spirulina levels were capable of effectively stimulating acid production of lactococci (Lc. lactis ssp. lactis Ha-2 and Lc. Lactis ssp. cremoris W-24). The cyanobacterial biomass, used at 0.1% to 0.8%, was found to significantly increase (P < 0.05) the rate of acid development by lactococci between h 6 and h 12 of the fermentation process (Molnár and others 2009). The addition of S. platensis caused a decline in pH values of yogurt samples. This decline was probably due to the stimulatory effect produced by the S. platensis biomass on the growth of L. bulgaricus, which was also supported by the higher viable cell counts of L. bulgaricus in algal yogurts in the 1st day of storage (Molnár and others 2005).

Slower mean pH drop rates were observed for the treatments constituting S. platensis. These treatments also showed significantly greater mean acidity increase rates (P < 0.05). In contrast, the control showed significantly lower mean acidity increase rates. Similar situations were observed for final acidity in the treatments. These characteristics can be attributed to the different buffering capacity effects of the treatments. Samples containing S. platensis exhibited higher buffering capacity. The greater the buffering capacity, the slower the pH drop and this stimulates acidification rate by starter bacteria because they are inhibited considerably later during fermentation (Beheshtipour and others 2012).

Considerable work has been reported on acid production of Enterococcus species in milk. In general, enterococci exhibit low milk acidifying ability (Giraffa 2003). Recent investigations on enterococci of dairy origin confirmed the poor acidifying capacity of these microorganisms in milk with only a small percentage of the strains showing a pH below 5.0 to 5.2 after 16 to 24 h of incubation at 37 °C (Andrighetto and others 2001; Durlu-Ozkaya and others 2001; Sarantinopoulos and others 2001). It was also demonstrated that E. faecalis is generally a stronger acidifier than E. faecium. A high acidifying potential in skim milk with a pH lowering to approximately 4.5 after 24 h of fermentation was observed for E. faecalis strains isolated from an Italian artisanal cheese (Giraffa and others 1993; Suzzi and others 2000). The specific enterococcal strain used in the trial of Giraffa and others (1993) showed good acidification properties by lowering the pH of control milks to between 5.06 and 5.15 after 22 h of fermentation at 37 °C. The acidity levels of 3.92 to 4.49 reached by the same E. faecium strain in Chlorella-supplemented milks under identical conditions were even lower than the value of 4.5 reported by Giraffa and others (1993) and Suzzi and other (2000) for the strong acidifier E. faecalis.

Lactobacillus plantarum proved to be a slightly poorer acidifier than E. faecium because the pH value of products ranged from 5.15 to 5.34 and from 4.62 to 5.10 in control and Spirulina-enriched samples, respectively, after 22 h of fermentation at 30 °C. However, similar to what was experienced with E. faecium, the addition of microalgal biomass had a significant stimulatory effect (P < 0.05) on L. plantarum throughout the entire fermentation process (Gyenis and others 2005).

It was found that there was a lower pH value in algal yogurt containing dried biomass of S. platensis than that of natural yogurt in the beginning of storage. Similarly, pH values in milk inoculated with the mixed culture of S. thermophilus and L. bulgaricus and containing S. platensis decreased more rapidly than in control samples during the 1st 3 h of fermentation (Varga and others 1999b; Varga and Szigeti 1998). Varga and others (2002) also determined a larger decrease in pH value of S. platensis-supplemented fermented ABT milk than in control ABT milk during storage. S. platensis-supplemented ABT fermented milk contained higher S. thermophilus counts than did the control ABT fermented milk after 6 wk of refrigerated storage. According to their results, yogurt supplemented with S. platensis had higher protein content compared to yogurt with no supplement. This was due to the high protein content of the microalga S. platensis (Varga and others 2002). De Caire and others (2000) found S. platensis to be a promoter for the growth of S. thermophilus in milk (De Caire and others 2000).

Table 1 shows selected publications on the effects of microalgal addition on pH and titratable acidity in yogurt. Figure 2 shows technological aspects of microalgal addition to probiotic fermented milks.

Table 1. Selected publications on the effects of microalgal addition on pH in fermented milks
Type of probioticExperiment conditionpH EPpH AFMicroalga addedReference
  1. EP, end of production; AF, after fermentation.

Lactococcus lactis ssp. lactis NCAIM B.21250 h/14 h–0.070.003 g/dm3 S. platensisMolnár and others (2009)
Lc. lactis ssp. lactis NCAIM B.21280 h/14 h–0.050.143 g/dm3 S. platensisMolnár and others (2009)
Lc. lactis ssp. lactis0 h/14 h–0.050.513 g/dm3 S. platensisMolnár and others (2009)
 var. diacetylactis NCAIM B.2122     
Lc. lactis ssp. lactis var. diacetylactis NCAIM B.21230 h/14 h–0.060.013 g/dm3 S. platensisMolnár and others (2009)
Lc. lactis ssp. lactis0 h/14 h–0.060.033 g/dm3 S. platensisMolnár and others (2009)
 var. diacetylactis NCAIM B.2126     
Lc. lactis ssp. lactis var. diacetylactis NCAIM B.21270 h/14 h–0.040.583 g/dm3 S. platensisMolnár and others (2009)
Lactococcus lactis ssp. cremoris ATCC 192570 h/14 h–0.050.063 g/dm3 S. platensisMolnár and others (2009)
Leuconostoc mesenteroides0 h/14 h–0.070.903 g/dm3 S. platensisMolnár and others (2009)
 ssp. cremoris NCAIM B.2120     
Leuconostoc mesenteroides0 h/14 h–0.050.103 g/dm3 S. platensisMolnár and others (2009)
 ssp. dextranicum NCAI B.1658     
LC. lactis ssp. lactis Ha-2, A0 h/24 h6.644.33A: with 0% S. platensisMolnár and others (2005)
LC. lactis ssp. lactis Ha-2, B0 h/24 h6.644.28B: with 0.1% S. platensisMolnár and others (2005)
LC. lactis ssp. lactis Ha-2, C0 h/24 h6.654.23C: with 0.3% S. platensisMolnár and others (2005)
LC. lactis ssp. lactis Ha-2, D0 h/24 h6.674.31D: with 0.5% S. platensisMolnár and others (2005)
LC. lactis ssp. cremoris A0 h/24 h6.634.25A: with 0% S. platensisMolnár and others (2005)
LC. lactis ssp. cremoris B0 h/24 h6.654.22B: with 0.1% S. platensisMolnár and others (2005)
LC. lactis ssp. cremoris C0 h/24 h6.664.22C: with 0.3% S. platensisMolnár and others (2005)
LC. lactis ssp. cremoris D0 h/24 h6.694.26D: with 0.5% S. platensisMolnár and others (2005)
LC. lactis ssp. cremoris E0 h/24 h6.644.29E: with 0.8% S. platensisMolnár and others (2005)
Enterococcus faecium A0 h/22 h6.314.04A: with12% total solid,Gyenis and others (2005)
     3 g/dm3 C. vulgaris 
 E. faecium B0 h/22 h6.334.06B: with 18% total solid,Gyenis and others (2005)
     3 g/dm3 C. vulgaris 
 E. faecium D0 h/22 h6.314.49D: with 30% total solid ,Gyenis and others (2005)
     3 g/dm3 C. vulgaris 
Lactobacillus plantarum A0 h/22 h6.484.62A: with 30% total solid ,Gyenis and others (2005)
     3 g/dm3 S. platensis 
 L. plantarum B0 h/22 h6.474.85B: with 30% total solid ,Gyenis and others (2005)
     3 g/dm3 S. platensis 
 L. plantarum C0 h/22 h6.474.86C: with 30% total solid ,Gyenis and others (2005)
     3 g/dm3 S. platensis 
 L. plantarum D0 h/22 h6.475.10D: with 30% total solid ,Gyenis and others (2005)
     3 g/dm3 S. platensis 
image

Figure 2. Technological aspects of microalgal addition in probiotic fermented milks.

Download figure to PowerPoint

Effects of Microalgal Supplementation on Viability of Probiotic Bacteria

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Yogurt or yogurt-like products have been used as the most popular carrier for incorporation of probiotic organisms. Unfortunately, most of the commercial products contain less probiotic bacteria than the minimum required, because these microorganisms grow slowly in milk and often show loss of viability during storage. In addition, the probiotic bacteria are sensitive to pH, lactic acid, hydrogen peroxide, and dissolved oxygen in fermented milk (Zhao and others 2006).

Various compositional and process factors significantly affect the viability of probiotic microorganisms in fermented milks including pH, titratable acidity, molecular oxygen, redox potential, hydrogen peroxide, bacteriocins, short-chain fatty acids, flavoring agents, microbial competitions, packaging materials and packaging conditions, rate and proportion of inoculation, step-wise/stage-wise fermentation, microencapsulation, milk solids nonfat content, supplementation of milk with nutrients, heat treatment of milk, incubation temperature, storage temperature, carbonation, addition of salt, sugar and sweeteners, cooling rate of the product, and scale of production (Mortazavian and others 2006; Champagne and Rastall 2009). pH is of the most critical factors decreases the viability of probiotic organisms in fermented milks (Tamime and others 2005; Korbekandi and others 2011).

Much effort has been made to improve the growth and survival of probiotic bacteria during storage. Some of the practices have been successful in improving survival of these bacteria in yogurt products. Substances such as oligosaccharides, sugar sources, and nonprotein nitrogen can improve the growth of probiotic bacteria. Vitamins, dextrin, and maltose stimulate the growth of bifidobacteria species in milk (Zhao and others 2006). Microalgae, including C. vulgaris and S. platensis, can increase viability of probiotic bacteria (Varga and others 2002; Gyenis and others 2005; Molnár and others 2009). In accordance with previous reports by various authors (Shirota and others 1964; Stengel 1970; Zielke and others 1978; Kurita and others 1979; Webb 1982), the substances responsible for the stimulatory properties of this cyanobacterial biomass were identified as adenine, hypoxanthine, and free amino acids.

It seems that co-culturing of microalgae and probiotics can stimulate growth and increase viability and acid production of probiotics in the products as well as in the gastrointestinal tract due to their alkaline character and presence of effective compounds such as adenine, hypoxanthine, and free amino acids (Gibson and Roberfroid 1995; Parada and others 1998). Some researchers have observed that growth of LAB in synthetic media was promoted by S. platensis extracellular product (De Caire and others 2000). Varga and others (1999b) reported that cyanobacterial biomass significantly stimulated growth and acid production of thermophilic dairy starter bacteria; therefore, it proved to be suitable for the cost-effective manufacture of novel functional fermented dairy foods (Varga and others 1999b).

As a general fact, the overgrowth of natural yogurt bacteria leads to the suppression of probiotics in fermented milks and the consequent viability reduction (Mortazavian and others 2005, 2006, 2007a, 2010, 2011; Shafiee and others 2010; Ahmadi and others 2011; Heydari and others 2011; Sadaghdar and others 2012). Therefore, the impact and control of microalgal addition on the viability of yogurt bacteria in fermented milks during fermentation and storage is rather important. De Caire and others (2000) studied the effect of a natural additive, a dry biomass from S. platensis, on the growth of LAB in milk. They grew S. thermophilus TH4, L. lactis C2, and L. delbrueckii YL1 with and without the addition of 3 mg of dry S. platensis/mL biomass. After 4 h, the LAB growth promotion by S. platensis, at pH 6.8, was 13.42% for C2, 9.29% for YL1, and 8.22% for TH4, compared with the controls. After 8 h, the increase was 3.46%, 9.73%, and 7.76% for C2, YL1, and TH4, respectively, and that is probably due to a decrease in the amount of the stimulatory factors. The 3 strains treated with Spirulina reached the stationary phase at 10 h and the counts remained the same up to 20 h, while the same strains without Spirulina addition grew more slowly and continued to grow up to 20 h, reaching the same value as the supplemented ones. The growth promotion of C2 was 27.3% after 4 h and for strain LO1, an increment of 22.8% was observed after 8 h. The tested LAB showed more growth in milk enriched with natural nutrients from the Spirulina, and they clearly responded to different extents according to the strain (De Caire and others 2000).

Parada and others (1998) found that addition of extracellular products obtained from a late log phase culture of S. platensis promoted the growth of some LAB. Hence, it was suggested that S. platensis could have a stimulatory effect on LAB by acting as a prebiotic factor. In this study, the addition of Spirulina filtrate was added to de man rogosa and sharpe agar and consequently, the bacterial growth was significantly stimulated for all the strains investigated. A similar effect was observed using enriched medium. The addition of zarrouk medium, treated the same as the Spirulina culture filtrates, did not change the extent of growth observed in the media prepared without extracellular products. In order to know the basic composition of the biomass of S. platensis and the cell-free culture medium filtrates, chemical analyses were performed. These chemical values were 7.3%, 1.7%, and 2.0%, before the growth of S. platensis, and 11.5%, 8.9%, and 1.3% after cultivating it to late log phase. Changes recorded in the above-mentioned parameters showed that S. platensis acted as a photoautotropic microorganism that consumes nitrogen from the culture medium and liberated exopolysaccharide and other compounds that could be responsible for the stimulatory effect on LAB (Parada and others 1998).

In Varga and other's study (1999a), S. platensis biomass showed no influence either on fermentation activity or on growth of B. bifidum or B. animalis when the milk was inoculated with a mixed culture of S. thermophilus and B. bifidum or B. animalis (Varga and others 1999a). Although the data on the viable cell counts of L. bulgaricus showed generally some fluctuations, in general, yogurt samples supplemented with S. platensis, had significantly higher viable counts of L. bulgaricus. Thus, supplementation with algal biomass significantly increased the viable counts of L. bulgaricus in both natural and probiotic yogurt. The stimulatory effect of the algal biomass on the survival of L. bulgaricus was noticeable throughout the storage period. This effect could be attributed to the presence of free amino acids, peptone, adenine, and hypoxanthine in the algal biomass because these nitrogenous substances are capable of significantly stimulating the growth and acid production of L. bulgaricus (Varga and others 1999a; Molnár and others 2005). In complementary research accomplished by Varga and others (1999b), there were significant differences in the viability of S. thermophilus between the yogurt samples with or without S. platensis. In general, higher viable counts of S. thermophilus were enumerated in yogurts containing no S. platensis during storage. Therefore, the addition of algal biomass significantly decreased the growth of S. thermophilus. The viable counts of S. thermophilus in all yogurt samples were enumerated above 8 log CFU/mL during 28 days (Varga and others 1999b). Furthermore, the Spirulina-supplemented fermented ABT milk contained significantly higher levels of viable bifidobacteria throughout the entire storage period than did the control product (Varga and others 2002). Varga and Szigeti (1998) enumerated minimum 8 log CFU/mL for viable counts of S. thermophilus in both natural and algal yogurt during storage at 4 °C. The survival rate of S. thermophilus was better than that of both L. bulgaricus and B. animalis. The viable counts of S. thermophilus were higher by 2 to 3 log orders than those for L. bulgaricus in yogurt samples (Varga and Szigeti 1998).

Molnár and others (2005) studied the effects of Spirulina biomass on single strains of mesophilic lactic acid bacteria. Used at the rate of 3 g/dm3, Spirulina significantly increased (P < 0.05) the acid production by various strains of mesophilic lactic acid bacteria. During the 1st 2 wk of refrigerated storage at 4 ± 2 °C, the Spirulina biomass significantly increased (P < 0.05) viability of mesophilic starter bacteria in the product. Because of its alkaline character and possession of considerable buffering capacity, Spirulina significantly stimulated the acid production and increased growth rates of some LAB during the fermentation process and even during the 1st week of storage. However, viability percentages declined slowly thereafter.

Gyenis and others (2005) studied the use of dried microalgal biomasses to stimulate acid production and growth of L. plantarum and E. faecium in milk. According to their results, acid production and growth of E. faecium and L. plantarum were stimulated significantly (P < 0.05) by C. vulgaris and S. platensis, respectively, in all culture media formulations used (Gyenis and others 2005). Their findings were consistent with those of Varga and others (1999b), who demonstrated that acid production and growth rates of thermophilic dairy starter cultures, such as S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, and B. bifidum, could be stimulated effectively by S. platensis biomass (Gyenis and others 2005).

Beheshtipour and others (2012) studied the effects of C. vulgaris and S. platensis addition on the viability of probiotic bacteria in yogurt and its biochemical properties. According to their results the viability of both probiotic bacteria (L. acidophilus LA-5 and B. lactis BB-12) was significantly and markedly greater in the treatments containing microalgae than the control. Also, the higher concentration of microalgae (from 0.25% to 1.0%) had greater viability of both probiotic bacteria at the end of fermentation and during refrigerated storage (Beheshtipour and others 2012). Table 1 shows selected publications on the effects of the addition of microalgae on the viability of lactic acid bacteria. Table 2 shows selected publications on the effects of microalgal addition on the viability of probiotics and lactic acid bacteria.

Table 2. Selected publications on the effects of microalgal addition on the viability of probiotics and lactic acid bacteria
  Viability Log CFU/mL  
ProbioticMicroalga addedControlMicroalga enrichedExperiment conditionReference
  1. EP, end of production; ES, end of storage; AF, after fermentation.

Streptococcus thermophilus3 mg/mL S. platensis9.039.17EPVarga and others (2002)
  8.999.27ES: 15 °C/18 d 
Lactobacillus acidophilus3 mg/mL S. platensis7.117.38EPVarga and others (2002)
  7.097.30ES: 15 °C/18 d 
Bifidobacteria3 mg/mL S. platensis6.196.36EPVarga and others (2002)
  5.135.33ES: 15 °C/18 d 
S. thermophilus3 mg/mL S. platensis9.039.17EPVarga and others (2002)
  8.648.86ES: 4 °C/42 d 
Lactobacillus acidophilus3 mg/mL S. platensis7.117.38EPVarga and others (2002)
  7.007.31ES: 4 °C/42 d 
Bifidobacteria3 mg/mL S. platensis6.196.36EPVarga and others (2002)
  4.694.86ES: 4 °C/42 d 
Lactococcus lactis ssp. Lactis NCAIM B.21283 mg/mL S. platensis∼770 h AFMolnár and others (2009)
  ∼8.58.512 h AF 
Lc. lactis ssp. lactis var. diacetylactis NCAIM B.21273 mg/mL S. platensis6.56.50 h AFMolnár and others (2009)
  8–8.58.512 h AF 
Lc. lactis ssp. cremoris ATCC 192523 mg/mL S. platensis770 h AFMolnár and others (2009)
  8–8.58.5–912 h AF 
S. thermophilus TH43 mg/mL S. platensis4.6024.6020 h AFDe Caire and others (2000)
  7.8517.89720 h AF 
Lactobacillus delbrueckii ssp. bulgaricus YL13 mg/mL S. platensis4.7854.7850 h AFDe Caire and others (2000)
  7.6437.75520 h AF 
L. Lactis ssp. Lactis C23 mg/mL S. platensis4.8924.8920 h AFDe Caire and others (2000)
  7.5317.49120 h AF 
Lactobacillus plantarum3 g/dm3 C. vulgaris6.786.880 h AFGyenis and others (2005)
  8.618.9822 h AF 
Enterococcus faecium3 g/dm3 C. vulgaris6.836.930 h AFGyenis and others (2005)
  8.729.0822 h AF 
Lactobacillus casei A1 mg/mL S. platensis3.663.660 h AFBhowmik and others (2009)
  6.006.5010 h AF 
L. casei B5 mg/mL S. platensis3.663.660 h AFBhowmik and others (2009)
  6.007.6610 h AF 
L. casei C10 mg/mL S. platensis3.663.660 h AFBhowmik and others (2009)
  6.009.0010 h AF 
Lactobacillus acidophilus A1 mg/mL S. platensis2.302.300 h AFBhowmik and others (2009)
  4.004.3310 h AF 
L. acidophilus B5 mg/mL S. platensis2.302.300 h AFBhowmik and others (2009)
  4.005.3310 h AF 
L. acidophilus C10 mg/mL S. platensis2.302.300 h AFBhowmik and others (2009)
  4.006.6610 h AF 
S. thermophilus A1 mg/mL S. platensis3.663.660 h AFBhowmik and others (2009)
  4.665.3310 h AF 
S. thermophilus B5 mg/mL S. platensis3.663.660 h AFBhowmik and others (2009)
  4.667.0010 h AF 
S. thermophilus C10 mg/mL S. platensis3.663.660 h AFBhowmik and others (2009)
  4.668.3310 h AF 

The addition of S. platensis increased the viable counts of L. bulgaricus in synthetic media (Parada and others 1998) and in milk (Varga and others 1999a; De Caire and others 2000). The higher counts of L. bulgaricus in probiotic yogurts containing microalgae could have resulted from the symbiosis between B. animalis and L. bulgaricus. Donkor and others (2006) reported that the presence of probiotic organisms, including bifidobacteria, increased proteolytic activity and improved the survival of L. bulgaricus in yogurt.

Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Addition of microalgae into fermented milks can change the sensory attributes, mostly undesirably, although there is not enough related information in the literature. In one study, as part of the product development process, 3 ranking tests were performed by 5, 11, and 12 sensory panelists, respectively, in an attempt to optimize the organoleptic properties of the final product. The samples were ranked according to the intensity of their sensory properties, with overall taste being the main ranking parameter. According to the results of the ranking tests done by panelists, optimum organoleptic properties were achieved in the product formulation prepared with the mixed culture of Lc. lactis subsp. lactis NCAIM B.2128 and Lc. lactis subsp. cremoris ATCC 19257, and supplemented with sucrose at 10%, Spirulina biomass at 0.3%, and strawberry-kiwifruit puree at 1.5% (Molnár and others 2009).

Beheshtipour and others (2012) reported that treatments with higher amounts of microalgae possessed weaker sensory acceptability for all sensory parameters compared to the control. S. platensis exhibited more unpleasant flavor compared to C. vulgaris, and the treatment with 1% S. platensis had the lowest flavor score. Addition of microalgae into the yogurt changed the color of this product to greenish or bluish based on the type and concentration of microalgae added. This characteristic was realized as an inappropriate sensory attribute (appearance) by the panelists.

Moreover, graininess caused by insoluble microalgal particles was recognized mostly in treatments with 1% microalgae. There were no considerable differences among the treatments from nonoral texture points of view. However, differences were remarkable from an oral texture standpoint. Treatments containing 1% microalgae had the lowest sensory score for oral texture and mouthfeel. Overall, treatments with C. vulgaris obtained higher sensory scores compared to S. platensis. There was no significant difference between the treatments containing 0.25% or 0.5% of both microalgae (Beheshtipour and others 2012).

Prakash and Kumari (2011) studied the preparation of low-fat and high-protein frozen yogurt enriched with papaya pulp and Spirulina with the objective to find out the optimum level of Spirulina that could be incorporated to obtain a better-quality frozen yogurt. It was observed that incorporation of Spirulina from 2% to 8%, before incubation with the addition of 10% papaya pulp, was the more acceptable. The frozen yogurt prepared with 6% Spirulina with 10% papaya pulp was found best on the basis of sensory attributes (score 8.6) compared to the rest of treatments tried in the study. Higher levels of Spirulina adversely affected sensory characteristics of frozen yogurt (Prakash and Kumari 2011).

Jeon (2006) investigated the effect of addition of Chlorella on the sensory properties of processed cheese. Processed cheeses were prepared with Chlorella (0.5% or 1.0% (w/w)) or without Chlorella (control). The cheeses were stored at 10 ± 1 °C. In comparison with the control cheese, quantitative descriptive analysis scores for color and mouthfeel were higher (P < 0.05) for processed cheeses with Chlorella. The processed cheese with 0.5% Chlorella was preferred to the 1.0% Chlorella and control cheeses (Jeon 2006).

For the purpose of making functional drinkable yogurt, new types of drinkable yogurts were prepared by Cho and others (2004) from skim milk with 0.25% Chlorella extract powder added and 2.5% to 10.0% chlorella extract (liquid) and then sensory properties were evaluated. The results of sensory evaluation of the drinkable yogurts containing Chlorella extract indicated that color, taste, aftertaste, and overall acceptability in the treatment with no addition of Chlorella extract had higher preference than all others. Also, sensory scores of the yogurt with 20% oligosaccharide added were significantly higher than other groups in aftertaste and overall acceptability. Gyenis and others (2005) reported that 3 g/dm3 of microalgal biomass was optimal regarding the sensory properties and cost (Cho and others 2004).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References

Microalgae as marine microorganisms have recently become popular as new source for both nutraceutical and pharmaceutical products. They have less complex biological systems compared to higher organisms. Undoubtedly, viability of probiotic bacteria during the fermentation process and subsequent refrigerated storage is a major concern in the production of probiotic yogurt or other milk products. The addition of microalgae such as Spirulina and Chlorella genera could raise the viability of probiotics in fermented dairy products like yogurt. However, this addition adversely affects the sensory attributes of the final product. Future investigations in this filed might be related to the addition of different types of microalgae into types of fermented milks other than yogurt, as well as improvement of sensory characteristics of the final products. Also, the effects of the addition of microalgae on in vivo viability of probiotics (instead of in-product evaluations) as well as on probiotics activity (not only assessing the viable bacterial count) is an important and uninvestigated topic that could be taken into consideration.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Fermented Milks as Probiotic Food Carrier
  5. The Spirulina and Chlorella Usage and Their Functional Ingredients
  6. Effects of Microalgae Supplementation on Acidification Rate in Fermenting Milk Containing Probiotic Bacteria
  7. Effects of Microalgal Supplementation on Viability of Probiotic Bacteria
  8. Effects of Microalgal Supplementation on Sensory Attributes of Fermented Milks
  9. Conclusions
  10. Acknowledgment
  11. References
  • Ahmadi E, Mortazavian AM, Fazeli MR, Ezzatpanah H, Mohammadi R, Dolatkhah-nejad MR, Bhadori Monfared A. 2012. The effects of inoculants variables on the physicochemical and organoleptic properties of doogh. Int J Dairy Technol 65(2):27481.
  • Andrighetto C, Knijff E, Lombardi A, Torriani S, Vancanneyt M, Kersters K, Swings J, Dellaglio F. 2001. Phenotypic and genetic diversity of enterococci isolated from Italian cheeses. J Dairy Res 68:30316.
  • Ayala F, Vargas T. 1987. Experiments on Spirulina culture on waste-effluent media at the pilot plant. Hydrobiologia 151/152:913.
  • Barrow C, Shahidi F. 2008. Marine nutraceuticals and functional foods. Boca Raton, Fla.: CRC Press. p 367403.
  • Barsanti L, Gualtieri P. 2006. Algae: anatomy, biochemistry, and biotechno-logy. New York, N.Y.: CRC Press.
  • Becker EW. 1984. Nutritional properties of microalgal potentials and constraints. In: Richmond A, editor. Handbook of microalgal mass culture. Boca Raton, Fla.: CRC Press, Inc. p 339408.
  • Beheshtipour H, Mortazavian AM, Haratian P, Khosravi Darani K. 2012. Effects of Chlorella vulgaris and Spirulina platensis addition on the viability of probiotic bacteria in yogurt and its biochemical properties. Eur J Food Res Technol DOI: 10.1007/s00217-012-1798-4.
  • Belasco W. 1997. Algae burgers for a hungry world? The rise and fall of Chlorella cuisine. Technol Cult (38)3:60834.
  • Belay A. 1997. Mass culture of Spirulina outdoors. The Earthrise Farms experience. In: Vonshak A, editor. Spirulina platensis (Arthrospira): physiology, cell biology and biotechnology. London: Taylor and Francis. p 13158.
  • Bhowmik D, Dubey J, Mehra S. 2009. Probiotic efficiency of Spirulina platensis – stimulating growth of lactic acid bacteria. American Eurasian J Agric Environ Sci 6(5):54649.
  • Canizares RO, Dominguez AR, Rivas L, Montes MC, Travieso L, Benitez F. 1993. Free and immobilized cultures of Spirulina maxima for swine waste treatment. Biotechnol Let 15:3216.
  • Chamorro G, Salzar M, Favila L, Bourges H. 1996. Farmacología y toxicología del alga Spirulina. Rev Invest Clin 48:38999.
  • Champagnem CP, Rastall RA. 2009. Some technological challenges in the addition of probiotic bacteria to foods. In: Charalampopoulos D, Rastall RA, editors. Prebiotics and probiotics science and technology. London: Springer. p 763806.
  • Cho EJ, Nam ES, Park SI. 2004. Keeping quality and sensory properties of drinkable yoghurt with added Chlorella extract. Korean J Food Nutr 17(2):12832.
  • Ciferri O. 1983. Spirulina, the edible microorganism. Microbiol Rev 47:55178.
  • Ciferri O, Tiboni O. 1985. The biochemistry and industrial potential of Spirulina. Ann Rev Microbiol 39:50326.
  • Cohen Z. 1997. The chemicals of Spirulina. In: Vonshak A, editor. Spirulina platensis (Arthrospira): physiology, cell-biology and biotechnology. London: Taylor and Francis. p 175204.
  • Cruz AG, Antunes AEC, Sousa ALOP, Faria JAF, Saad SMI. 2009a. Ice cream as a probiotic food carrier. Food Res Int 42:12339.
  • Cruz AG, Buriti FCA, de Souza CHB, Faria JAF, Saad SMI. 2009b. Probiotic cheese: health benefits, technological and stability aspects. Trends Food Sci Technol 20:34454.
  • Cruz AG, Candena RS, Walter EHM, RS, Mortazavian AM, Granato D, Faria JAF, Bolini MA. 2010. Sensory analysis: relevance for probiotic, prebiotic and symbiotic product development. Comp Rev Food Sci Food Safety 9:35873.
  • Cruz AG, Castro WF, Faria JAF, Bogusz S, Granato D, Celeguini RMS, Lima-Pallone J, Godoy HT. 2012a. Glucose oxidase: a potential option to decrease the oxidative stress in stirred probiotic yogurt. LWT Food Sci Technol 47(2):5125.
  • Cruz AG, Castro WF, Faria JAF, Lollo PCB, Amaya-Farfán J, Freitas MQ, Rodrigues D, Oliveira CAF, Godoy HT. 2012b. Probiotic yogurts manufactured with increased glucose oxidase levels: postacidification, proteolytic patterns, survival of probiotic microorganisms, production of organic acid and aroma compounds. J Dairy Sci 95(5):22619.
  • Dave RI, Shah NP. 1997. Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter cultures. Int Dairy J 7:3141.
  • De Caire GZ, Parada JL, Zaccaro MC, de Cano MMS. 2000. Effect of Spirulina platensis biomass on the growth of lactic acid bacteria in milk. World J Microbiol Biotechnol 16:5635.
  • Dillon JC, Phan PA. 1993. Spirulina as a source of proteins in human nutrition. In: Doumengue F, Durand-Chastel H, Toulemont A, editors. Spiruline algue de vie. Musée Océanographique. Bulletin de l'Institut Océanographique Monaco. Numéro special 12:1037.
  • Doumenge F, Durand-Chastel E, Toulemont A. 1993. Spirulina algue de vie. Bull Inst Oceanogr Monaco. 222 pp.
  • Dubacq JP, Pham-quoc K. 1993. Biotechnology of Spirulina lipids: a source of gamma-linolenic acid. In: Doumengue F, Durand-Chastel H, Toulemont A, editors. Spiruline algue de vie. Musée Océanographique. Bulletin de l'Institut Océanographique Monaco. Numéro spécial 12:5964.
  • Durlu-Ozkaya F, Xanthopoulos V, Tunail N, Litopoulou-Tzanetaki E. 2001. Technologically important properties of lactic acid bacteria isolates from Beyaz cheese made from raw ewes’ milk. J Appl Microbiol 9:86170.
  • Fox RD. 1986. Algaculture: la Spirulina, UN espoir pour le monde de la faim, Edisude, France.
  • Fox D. 1993. Health benefits of Spirulina and proposal for a nutrition test on children suffering from kwashiorkor and marasmus. In: Doumengue F, Durand-Chastel H, Toulemont A, editors. Spiruline algue de vie. Bulletin de l'Institut Océanographique Monaco, Musée Océanographique. Numéro spécial 12:17985.
  • Fuller R. 1989. Probiotics in man and animals. J Appl Bacteriol 66:36578.
  • Gibson GR, Roberfroid MB. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 125:140112.
  • Giraffa G. 2003. Functionality of enterococci in dairy products. Int J Food Microbiol 88:21522.
  • Giraffa G, Gatti M, Carminati D, Neviani E. 1993. Biochemical and metabolic characteristics of strains belonging to Enterococcus genus isolated from dairy products. Proceedings of the Congress on Biotechnology and Molecular Biology of Lactic Acid Bacteria for the Improvement of Foods and Feeds Quality, Naples, February 2324.
  • Gismondo MR, Drago L, Lombardi A. 1999. Review of probiotics available to modify gastrointestinal flora. Int J Antimicrob Agent 12:28792.
  • Gomes AMP, Malcata FX, Klaver FAM, Grande HJ. 1995. Incorporation and survival of Bifidobacterium sp. Strain Bo and Lactobacillus acidophilus strain Ki in a cheese product. Neth Milk Dairy J 49:7195.
  • Granato D, Branco GF, Nazzaro F, Cruz AG, Faria JAF. 2010a. Functional foods and nondairy probiotic food development: trends, concepts, and products. Comp Rev Food Sci Food Safety 9(3):292302.
  • Granato Dl, Branco GF, Cruz AG, Faria JAF, Shah NP. 2010b. Probiotic dairy products as functional foods. Comp Rev Food Sci Food Safety 9(5):45570.
  • Gueimonde M, Delgado S, Mayo B, Ruas-Madiedo P,Margolles A, de los Reyes-Gavil C. 2004. Viability and diversity of probiotic Lactobacillus and Bifidobacterium populations included in commercial fermented milks. Food Res Int 37:83950.
  • Gyenis B, Szigeti J, Molnár N, Varga L. 2005. Use of dried microalgal biomasses to stimulate acid production and growth of Lactobacillus plantarum and Enterococcus faecium in milk. Acta Agraria Kaposváriensis 9(2):539.
  • Hayashi K. 1996. Calcium-Spirulan, an inhibitor of enveloped virus replication, from a blue-green alga Spirulina. J Nat Prod 59:837.
  • Henrikson R. 1994. Microalga Spirulina, superalimento del futuro. Ronore Enterprises. 2nd ed. Barcelona, Spain: Ediciones Urano. p 222.
  • Heydari S, Mortazavian AM, Ehsani MR, Mohammadifar MA, Ezzatpanah H, Sohrabvandi S. 2011. Biochemical, microbiological and sensory characteristics of probiotic yogurt containing various prebiotic or fiber compounds. Italian J Food Sci 23:15363.
  • Hoier E. 1992. Use of probiotic starter cultures in dairy products. Food Australia 44:41820.
  • Holzaspfel WH, Schillinger U. 2001. Introduction to pre- and probiotics. Food Res Int 35:10916.
  • Janczyk P, Franke H, Souffrant WB. 2007. Nutritional value of Chlorella vulgaris: effects of ultrasonication and electroporation on digestibility in rats. Animal Feed Sci Technol 132:1639.
  • Jeon JK. 2006. Effect of Chlorella addition on the quality of processed cheese. J Korean Soc Food Sci Nutr 35(3):3737.
  • Jeong H, Kwon HJ, MK. 2009. Hypoglycemic effect of Chlorella vulgaris intake in type 2 diabetic Goto-Kakizaki and normal Wistar rats. Nutr Res Pract 3(1):2330.
  • Kailasapathy K, Rybka S. 1997. Lactobacillus acidophilus and Bifidobacterium spp.—their therapeutic potential and survival in yogurt. Aust J Dairy Technol 52:2833.
  • Karimi R, Mortazavian AM, Cruz AG. 2010. Viability of probiotics in cheese during production and storage. Dairy Sci Technol 91:283308.
  • Klaver FAM, Kingma F, Weerkamp AH. 1993. Growth and survival of bifidobacteria in milk. Neth Milk Dairy J 47:15164.
  • Korbekandi H, Mortazavian AM, Iravani S. 2011. Technology and stability of probiotics in fermented milks. In: Probiotic and prebiotic foods: technology, stability and benefits to human health. Shah N, editor. New York, N.Y. Nova Science Publishers, Inc.
  • Kreitlow S, Mundt S, Lindequist U. 1999. Cyanobacteria — a potential source of new biologically active substances. J Biotechnol 70:613.
  • Kurita H, Tajima O, Fukimbara T. 1979. Isolation and identification of nucleosides in Chlorella extract. J Agr Chem Soc 53(4):1313.
  • Lacaz R, Nascimento E. 1990. Produçáo de biomassa de Spirulina máxima para alimentaçáo humana e animal. Rev Microbiol 21:8597.
  • Marth EH, Steele JL. 1998. Applied dairy microbiology. New York, N.Y. Marcel Dekker, Inc.
  • Mendes RL, Nobre BP, Cardoso MT, Pereira AP, Palavra AF. 2003. Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Inorg Chim Acta 356:32834.
  • Merchant RE, Andre CA. 2001. A review of recent clinical trials of the nutritional supplement Chlorella pyrenoidosa in the treatment of fibromyalgia, hypertension, and ulcerative colitis. Altern Ther Health Med 7(3):7991.
  • Mohammadi R, Mortazavian A M. 2010. Technological aspects of prebiotics in probiotic fermented milks. Food Rev Int 27:192212.
  • Mohammadi R, Mortazavian AM, Khosrokhavar R, Cruz AG. 2011. Probiotic ice cream: viability of probiotic bacteria and sensory properties. Annal Microbiol 61:41124
  • Molnár N, Gyenis B, Varga L. 2005. Influence of a powdered Spirulina platensis biomass on acid production of lactococci in milk. Milchwissenschaft 60(4):3802
  • Molnár N, Sipos-Kozma Zs, Tóth Á, Ásványi B, Varga L. 2009. Development of a functional dairy food enriched with Spirulina (Arthrospira platensis). Tejgazdaság 69(2):1522
  • Mortazavian AM, Ehsani MR, Mousavi SM, Reinheimer J, Emamdjomeh Z, Sohrabvandi S. 2005. Preliminary investigation on the combined effect of heat treatment and incubation temperature on the viability of the probiotic microorganisms in freshly made yoghurt. Int J Dairy Technol 59:811.
  • Mortazavian AM, Sohrabvandi S, Mousavi SM, Reinheimer JA. 2006. Combined effects of temperature-related variables on the variables on the viability of probiotics in yogurt. Aust J Dairy Technol 61:24852.
  • Mortazavian AM, Ehsani MR, Mousavi SM, Rezaei K, Sohrabvandi S, Reinheimer JA. 2007a. Effect of refrigerated storage temperature on the viability of probiotic micro-organisms in yogurt. Int J Dairy Technol 60(2):1237.
  • Mortazavian AM, Ehsani MR, Sohrabvandi S, Reinheimer JA. 2007b. MRS-bile agar: its suitability for the enumeration of mixed probiotic cultures in cultured dairy products. Milchwissenschaft 62(3):2702.
  • Mortazavian AM, Ehsani MR, Mousavi SM, Sohrabvandi S, Reinheimer JA. 2007c. Effect of refrigerated storage temperature on the viability of probiotic micro-organisms in yoghurt. Int J Dairy Technol 59:1237.
  • Mortazavian AM, Razavi SH, Ehsani MR, Sohrabvandi S. 2007d. A review: principles and methods of microencapsulation of probiotics. Iran J Biotechnol 5:118.
  • Mortazavian AM, Ehsani SH, Razavi MR, Mousavi SM, Sohrabvandi S, Reinheimer JA. 2008. Effect of microencapsulation of probiotic bacteria with calcium alginate on cell stability during the refrigerated storage period in Iranian yogurt drink (doogh). Milchwissenschaft. 63:2625.
  • Mortazavian, AM, Rezaei K, Sohrabvandi S. 2009. A review: application of advanced instrumental methods for yogurt analysis. Crit Rev Food Sci Nutr 49:15363.
  • Mortazavian AM, Khosrokhavar R, Rastgar H. 2010. Effects of dry matter standardization order on biochemical and microbiological characteristics of doogh (Iranian fermented milk drink). Italian J Food Sci 22:98104.
  • Mortazavian AM, Ghorbanipour S, Mohammadifar MA, Mohammadi M. 2011. Biochemical properties and viable probiotic population of yogurt at different bacterial inoculation rates and incubation temperatures. Philipp Agric Scientist 94:1116.
  • Nakano S, Noguchi T, Takekoshi H, Suzuki G, Nakano M. 2005. Maternal-fetal distribution and transfer of dioxins in pregnant women in Japan, and attempts to reduce maternal transfer with Chlorella (Chlorella pyrenoidosa) supplements. Chemosphere 61(9):124455.
  • Nakasawa Y, Hosono A. 1992. Functions of fermented milk. Challenges for the health sciences. London, England: Elsevier Applied Science.
  • Neuringer M, Connor WE. 1986. N-3(W3) fatty acids in the brain and retina. Evidence for their essentiality. Nutr Rev 44(9):28594.
  • Nighswonger BD, Brashears MM, Gilliland SE. 1996. Viability of Lactobacillus acidophilus and Lactobacillus casei in fermented milk products during refrigerated storage. J Dairy Sci 79:2129.
  • Nobakhti AR, Ehsani MR, Mousavi SM, Mortazavian AM. 2008. Influence of lactulose and Hi-maize addition on viability of probiotic microorganisms in freshly made symbiotic fermented milk drink. Milchwissenschaft 63:4279.
  • Othes S, Pire R. 2001. Fatty acid composition of Chlorella and Spirulina microalgae species. J AOAC Int 84:170814.
  • Oxa P, Rios J. 1998. Proyecto de desarrollo técnico-económico en la utilización de la biotecnología microalgal para el cultivo de Spirulina platensis. Trabajo de Grado. Arica, I Región de Tarapacá, Universidad Arturo Prat, Iquique, Chile.
  • Parada JL, de Caire GZ, de Mulé MCZ, de Cano MMS. 1998. Lactic acid bacteria growth promoters from Spirulina platensis. Int J Food Microbiol 45:2258.
  • Prakash DR, Kumari P. 2011. Preparation of low-fat and high-protein frozen yoghurt enriched with papaya pulp and Spirulina. Trends Biosci 4(2):1824.
  • Pugh N, Ross SA, Elsohly HN, Elsohly MA, Pasco DS. 2001. Isolation of three weight polysaccharide preparations with potent immunostimulatory activity from Spirulina platensis, Aphanizomenon flos-aguae and Chlorella pyrenoidosa. Planta Med 67:73742.
  • Ravula RR, Shah NP. 1998. Effect of acid casein hydrolysate and cysteine on the viability of yogurt and probiotic bacteria in fermented frozen dairy desserts. Aust J Dairy Technol 53:1759.
  • Richmond A, 1984. Spirulina. In: Borowitzka MA, Borowitzka LJ, editors. Micro-algal biotechnology. Cambridge, UK: University Press. p85121.
  • Richmond A. 1992. Mass culture of cyanobacteria. In: Mann N, Carr N, editors. Photosynthetic prokaryotes. 2nd ed. New York, N.Y. and London: Plenum Press. p 181210.
  • Riordan KO, Fitzgerald GF. 1998. Evaluation of bifidobacteria for the production of antimicrobial compounds and assessment of performance in cottage cheese at refrigeration temperature. J Appl Microbiol 85:10314.
    Direct Link:
  • Saarela M, Mogensen G, Fonden R, Matto J, Mattila-Sandholm T. 2000. Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84:197215.
  • Sadaghdar Y, Mortazavian AM, Ehsani MR. 2012. Survival and activity of five probiotic lactobacilli strains in two types of flavored fermented milk. Food Sci Biotechnol 21(1):1517.
  • Salminen S, Wright AV. 1998. Lactic acid bacteria, microbiology functional aspects. New York, N.Y.: Marcel Dekker, Inc.
  • Sánchez M, Bernal-Castillo J, Rozo C, Rodríguez I. 2003. Spirulina (Arthrospira): an edible microorganism. Revista Universitas Scientiarum 8:724.
  • Sanders ME. 1999. Probiotics. Food Technol 53:6775.
  • Sarantinopoulos P, Andrighetto C, Georgalaki MD, Rea MC, Lombardi A, Cogan T M, Kalantzopoulos G, Tsakalidou E. 2001. Biochemical properties of enterococci relevant to their technological performance. Int Dairy J 11:62147.
  • Saxena PN, Ahmad MR, Shyan R, Amla DV. 1983. Cultivation of Spirulina in sewage for poultry feed. Experientia 39:107783.
  • Scheffler J, 2007. Underwater Habitats. Illumin 9 (4):19.
  • Schwartz J, Shklar G. 1987. Regression of experimental hamster cancer by beta-carotene and algae extracts. J Oral Maxillofac Surg 45:5105.
  • Schwartz R, Hirsch C, Sesin D, Flor J, Chartrain M, Frontling R, Harris G, Salvatore M, Liesch J, Yudin K. 1990. Pharmaceuticals from cultured algae. J Ind Microbiol Biotech 5:11324.
  • Sedef Nehir El, Simsek S. 2012. Food technological applications for optimal nutrition: an overview of opportunities for the food industry. Comp Rev Food Sci Food Safety 11(1):212.
  • Shafiee G, Mortazavian AM, Mohammadifar MA, Koushki MR, Mohammadi AR, Mohammadi R. 2010. Combined effects of dry matter content, incubation temperature and final pH of fermentation on biochemical and microbiological characteristics of probiotic fermented milk. Afr J Microbiol Res 4:126574.
  • Shah NP. 2000. Probiotic bacteria: enumeration and survival in dairy foods. J Dairy Sci 83:894907.
  • Shah NP. 2001. Functional foods from probiotics and prebiotics. Food Technol 55(11):4653.
  • Shekharam K, Ventakaraman L, Salimath P. 1987. Carbohydrate composition and characterization of two unusual sugars from the blue-green algae Spirulina platensis. Phytochemistry 26:22679.
  • Shimamatsu H. 2004. Mass production of Spirulina, an edible microalga. Hydrobiologia 512:3944.
  • Shirota M, Nagamatsu N, Takechi Y. 1964. Method for cultivating lactobacilli. U.S. Pat. No. 3,123,538.
  • Singh SP, Tiku AB, Kesavan PC. 1995. Post-exposure radioprotection by Chlorella vulgaris (E-25) in mice. Indian J Exp Biol 33(8):6125.
  • Sohrabvandi S, Razavi SH, Mousavi SM, Mortazavian AM. 2010. Viability of probiotic bacteria in low-alcohol- and non-alcoholic beer during refrigerated storage. Philipp Agric Scientist 93:248.
  • Spolaore P, Joannis-Cassan C, Duran E, Isambert A. 2006. Commercial applications of microalgae. J Biosci Bioen 101(2):8796.
  • Stengel E. 1970. Anlagentypen und Verfahren der technischen Algenmassenproduktion. Ber Dtsch Bot Ges 83:589606.
  • Suzzi G, Lombardi A, Lanorte MT, Caruso M, Andrighetto C, Gardini F. 2000. Characterization of autochthonous enterococci isolated from Semicotto Caprino cheese, a traditional cheese produced in Southern Italy. J Appl Microbiol 89:26774.
  • Takekoshi H, Suzuki G, Chubachi H, Nakano M. 2005. Effect of Chlorella pyrenoidosa on fecal excretion and liver accumulation of polychlorinated dibenzo-p-dioxin in mice. Chemosphere 59(2):297304.
  • Tamime AY, Saarela M, Korslund Sondergaard A, Mistry VV, Shah NP. 2005. Production and maintenance of viability probiotics microorganism in dairy products. In: Tamime AY, editor. Probiotic dairy products. London, UK: Blackwell Publishing Ltd. p 3997.
  • Tokus Oglu O, Unal MK. 2003. Biomass nutrient profiles of three microalgae: Spirulina platensis, Chlorella vulgaris, and Isochrisis galbana. Food Chem Toxicol 68(4):11448.
  • Toyomizu M, Sato K, Taroda H, Kato T, Akiba Y. 2001. Effects of dietary supplementation of Spirulina on meat color muscle of broiler chickens. Br Poul Sci 42:197202.
  • Valdivia VB, Butrón RO, Reynoso MP, Garcia AH, Europa EC. 2011. Chlorella vulgaris administration prevents HgCl2-caused oxidative stress and cellular damage in the kidney. J Appl Phycol 23:535.
  • Varga L, Szigeti J. 1998. Microbial changes in natural and algal yoghurts during storage. Acta Aliment Hung 27(2):12735.
  • Varga L, Szigeti J, Ördög V. 1999a. Effect of a Spirulina platensis biomass enriched with trace elements on combinations of starter culture strains employed in the dairy industry. Milchwissenschaft 54(5):2478.
  • Varga L, Szigeti J, Ördög V. 1999b. Effect of a Spirulina platensis biomass and that of its active components on single strains of dairy starter cultures. Milchwissenschaft 54:18790.
  • Varga L, Szigeti J, Kovács R, Földes T, Buti, S. 2002. Influence of a Spirulina platensis biomass on the microflora of fermented ABT milks during storage. J Dairy Sci 85:10318.
  • Webb LE. 1982. Detection by Warburg manometry of compounds stimulatory to lactic acid bacteria. J Dairy Res 49:47986.
  • Wood BJB. 1992. The lactic acid bacteria in health and disease. London, England: Elsevier Applied Science.
  • Yamaguchi K. 1996. Recent advances in microalgal bioscience in Japan, with special reference to utilization of biomass and metabolites: a review. J Appl Phycol 8:487502.
  • Zare F, Orsat V, Champagne C, Simpson B J, Boye JI. 2011. Microbial and physical properties of probiotic fermented milk supplemented with lentil flour. Food Res Int 44:24828.
  • Zhao QZ, Wang JS, Zhao MM, Jiang YM, Chun C. 2006. Effect of casein hydrolysates on yogurt fermentation and texture properties during storage. Food Technol Biotechnol 44(3):42934.
  • Zielke H, Kneifel H, Webb LE, Soeder CJ. 1978. Stimulation of lactobacilli by an aqueous extract of the green alga Scenedesmus acutus 276–3a. Eur J Appl Microbiol Biotechnol 6:7986.