Influence of a Spirulina platensis Biomass on the ... - ScienceDirect

J. Dairy Sci. 85:1031–1038  American Dairy Science Association, 2002.

Influence of a Spirulina platensis Biomass on the Microflora of Fermented ABT Milks During Storage (R1) L. Varga,* J. Szigeti,* R. Kovaćs,* T. Fo¨ldes,† and S. Buti* *Department of Food Technology and Microbiology and †Department of Dairy Science, Faculty of Agricultural and Food Sciences, University of West Hungary, 9200 Mosonmagyaro´va´r, Hungary

ABSTRACT

INTRODUCTION

The objective of this research was to investigate the effect of a cyanobacterial (Spirulina platensis) biomass on the microflora of a probiotic fermented dairy product during storage at two temperatures. Spirulinaenriched and control (plain) fermented acidophilusbifidus-thermophilus (ABT) milks were produced using a fast fermentation starter culture (ABT-4) as the source of Lactobacillus acidophilus (A), bifidobacteria (B), and Streptococcus thermophilus (T). Incubation took 6 h at 40°C. As for the cyanobacterial product, the S. platensis biomass was added to the process milk during stirring at pH 4.5 to 4.6. Thereafter, the ABTtype fermented milks were cooled to 25°C in ice water, filled into sterile, tightly capped centrifuge tubes, further cooled at 4°C for 24 h, and then stored either at 15°C for 18 d or at 4°C for 42 d. Microbiological analyses and acidity measurements were performed at regular intervals. Our results showed that the counts of the starter organisms were satisfactory during the entire storage period at both temperatures applied in this research. The S. platensis biomass had a beneficial effect on the survival of ABT starter bacteria regardless of storage temperature. Postacidification was observed at 15°C, whereas pH remained stable during refrigerated storage at 4°C. The abundance of bioactive substances in S. platensis is of great importance from a nutritional point of view because thus the cyanobacterial biomass provides a new opportunity for the manufacture of functional dairy foods. (Key words: Spirulina platensis, bifidobacteria, Lactobacillus acidophilus, Streptococcus thermophilus)

Bifidobacteria and certain Lactobacillus species have recently received attention as probiotic organisms maintaining a healthy equilibrium between the populations of beneficial and potentially harmful microorganisms in the gastrointestinal tract. They have been associated with health-promoting effects (Lankaputhra et al., 1996), and thus have been incorporated into a wide range of dairy foods (Shin et al., 2000). The probiotic properties of Bifidobacterium spp. and Lactobacillus acidophilus include prevention of traveler’s diarrhea (Alm, 1991), reduction of diarrhea and rotavirus infection in infants (Saavedra et al., 1994), prevention of constipation in elderly people (Alm, 1991), contribution to a faster recolonization of the intestinal microflora after administration of antibiotics (Black et al., 1991), improvement in lactose intolerance (Lin et al., 1991), reduction of cholesterol level in the blood (Agerbaek et al., 1995), stimulation of the immune system (Schiffrin et al., 1995), and improvement in defense against cancer (Krishnakumar and Gordon, 2001). It is our belief that consumers would need to ingest considerably less medicine and artificially produced vitamin and mineral supplements if fermented milks were enriched with vitamins, proteins, essential fatty acids, and trace elements of natural origin. A simple way of attaining this goal is the use of cyanobacteria in manufacture of cultured dairy products (Varga and Szigeti, 1998). Cyanobacteria belonging to prokaryotic algae are more closely related to bacteria than to other (eukaryotic) algae. They differ from photosynthetic bacteria in their photosynthetic pigments and in their ability to produce oxygen. Spirulina platensis is a planktonic cyanobacterium that forms massive populations in tropical and subtropical water bodies characterized by high levels of carbonate and bicarbonate and pH values of up to 11 (Tomaselli, 1997). The dried biomass of S. platensis typically contains 3 to 7% moisture, 55 to 60% protein, 6 to 8% lipids, 12 to 20% carbohydrate, 7 to 10% ash, 8 to 10% fiber, 1 to 1.5% chlorophyll a,

Abbreviation key: ABT = acidophilus-bifidus-thermophilus, DVS = direct vat set, MPN = most probable number.

Received August 16, 2001. Accepted December 19, 2001. Corresponding author: L. Varga; e-mail: [email protected].

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and a wide range of vitamins (Belay, 1997; Cohen, 1997; Vonshak, 1997a). Spirulina platensis is especially rich in proteins. The proteins with the highest economic potential are the biliproteins (e.g., c-phycocyanin and allophycocyanin), which are water-soluble blue pigments. The protein fraction may have a phycocyanin content of up to 20% (Cohen, 1997). Fatty acid composition is largely influenced by environmental conditions. Spirulina platensis can be characterized by about 45 to 50% saturated and 50 to 55% unsaturated fatty acids. Up to 30% of fatty acids is gammalinolenic acid, a rare polyunsaturated fatty acid claimed to have medicinal properties. Beneficial Spirulina strains and an efficient processing procedure should yield biomass with at least 1% gamma-linolenic acid (Cohen, 1997; Vonshak, 1997a). Since the late 1970s, S. platensis has been marketed and consumed as a safe human food and has been approved for human nutrition by many governments, health agencies, and associations of some 80 countries, including the United States and Hungary. Based on 30 yr of safety and quality research, several countries and organizations have established Spirulina quality and safety standards. More than 70% of the current Spirulina market is for human consumption, mainly as health food. The total annual production of food-grade Spirulina biomass is estimated to be approximately 1000 to 1500 tonnes (Belay, 1997; Vonshak, 1997b). Regulatory authorities around the world are looking for assurance that a probiotic product can deliver viable starter organisms at sufficient numbers to the large intestine in order to provide a benefit to the consumer. Concentrations of at least 106 to 107 cfu/g should be present at the time of consumption if a health claim is to be made (Gla¨ser, 1992; Krishnakumar and Gordon, 2001). Food regulations in Hungary stipulate that probiotic fermented milks must, until the end of the expiration date, have colony counts for starter organisms in the region of 107 cfu/g of product (Codex Alimentarius Hungaricus Commission, 1999). In the United States, there is no minimum live bacteria count required, except in the states of California and Oregon, where 2.0 × 106 cfu/ml are required for probiotic dairy products (Krishnakumar and Gordon, 2001). The purpose of this study was to investigate the effect of a cyanobacterial biomass on the characteristic microbial flora and spoilage organisms of fermented acidophilus-bifidus-thermophilus (ABT) milks during storage at 15°C and at 4°C. MATERIALS AND METHODS Raw Material Commercially available UHT milk (4 L) containing (per liter) 36 g of fat, 34 g of protein, 47 g of lactose, Journal of Dairy Science Vol. 85, No. 5, 2002

and 7 g of ash was used, to which 10 g of skim milk powder per liter was added. This raw material, measured into two jars (2 L), was heated to 90°C and held for 10 min before being cooled to inoculation temperature to insure adequate whey protein denaturation (Kessler, 1988). Starter Culture The ABT-4 culture (Chr. Hansen A/S, Hørsholm, Denmark) was kindly supplied in freeze-dried direct vat set (DVS) form by the Hungarian Dairy Research Institute, Inc. (Mosonmagyaro´va´r, Hungary). It was used for direct inoculation of process milks because DVS cultures need no activation or other treatment prior to use. Spirulina platensis Biomass The S. platensis Hau biomass was obtained from the Institute of Cereal Processing, Inc. (Bergholz-Rehbru¨cke, Germany). It contained (per kilogram) 941 g of DM, 576 g of protein, 111 g of total lipids, and 114 g of ash. Previous work (Springer et al., 1998) indicated that 3 g/L of cyanobacterial biomass was optimal in regards to sensory properties and cost. Manufacture and Storage of Fermented ABT Milks The ABT-4 freeze-dried DVS culture was added to the heat-treated milks cooled to 42°C at the rate of 0.2 U/L, corresponding to 2% (vol/vol) conventional bulk starter. Incubation took 6 h at 40°C. In the case of the cyanobacterial product, the S. platensis biomass was added to the process milk (2 L) during stirring at pH 4.5 to 4.6. Thereafter, the Spirulina-enriched and control fermented ABT milks were both cooled to 25°C in ice water and were then filled into 40 sterile, tightly capped centrifuge tubes (50 ml). Thus, 80 units of product were manufactured altogether. After 24 h of cooling at 4°C (d 0), one half of the samples of both products was placed into a cooling and heating thermostat at 15°C, whereas the other half was stored at refrigeration temperature (4°C). The entire experimental program was repeated three times. Microbiological Analysis Three cyanobacterial and three control samples were taken at each sampling time, i.e., after 0, 3, 6, 9, 12, 15, and 18 d of storage at 15°C and after 0, 7, 14, 21, 28, 35, and 42 d of storage at 4°C. On d 0, only three Spirulina-enriched samples and three controls

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were taken altogether instead of 2 × 3 because, at this stage, there was not yet any difference between the two batches. Samples were aseptically removed from centrifuge tubes and diluted by mixing 10 ml with 90 ml of 0.1% peptone water. Further dilutions were made as required. The pour-plate method was used to enumerate organisms except for coliforms and Escherichia coli, for which the more appropriate most probable number (MPN) technique was employed. Streptococcus thermophilus. M17 agar (Oxoid Ltd., Basingstoke, UK) was used to enumerate S. thermophilus. The pH of the medium was 6.9 ± 0.1. The inoculated plates were incubated at 37°C for 48 h under aerobic conditions. Streptococcus thermophilus formed lenticular colonies with a diameter of 1 to 2 mm. Colony-forming units (cfu), expressed as log per milliliter, were used to report survival of streptococci. Lactobacillus acidophilus. MRS-Maltose agar (Chr. Hansen A/S, 1995) with pH 6.2 ± 0.1 was used for enumeration of L. acidophilus. The plates were incubated at 37°C for 72 h. Anaerobic culture jars (2.5 L) were employed to generate anaerobic conditions, atmospheric oxygen being absorbed by means of AnaeroGen AN 25 sachets (Oxoid). The counts were expressed as log cfu/ml. The lactobacilli identified on the basis of colonial type were confirmed by microscopic examination. Lactobacillus acidophilus were Grampositive rods with rounded ends. Bifidobacterium spp. Nalidixic acid (0.030 g), neomycin sulfate (0.200 g), lithium chloride (0.600 g), and paromomycin sulfate (0.250 g), all obtained from Sigma Chemical Co. (St. Louis, MO), were suspended in distilled water (100 ml), and then sterilized by filtering through Millipore filters (Millipore Corporation, Bedford, MA) with pore diameter of 0.22 µm. The pH of the solution was adjusted to 7.3 ± 0.1 with 0.1 M sodium hydroxide (NaOH) before sterilization. A 5-ml aliquot of this antibiotic solution was added to 100 ml of MRS agar (pH 6.2 ± 0.1; Oxoid) immediately before use. The culture medium thus prepared was used for the enumeration of bifidobacteria. The plates were incubated at 37°C for 5 d. Anaerobic conditions were generated using anaerobic culture jars (2.5 L) and AnaeroGen AN 25 sachets (Oxoid). The counts were expressed as log cfu/ml. The bifidobacteria colonies identified were irregularly shaped or lenticular, and were corroborated by observation under a microscope. Yeasts and molds. YGC agar (Merck KGaA, Darmstadt, Germany) was used to enumerate yeasts and molds. The pH of the medium was 6.6 ± 0.1. The inoculated plates were incubated aerobically at 25°C for 5 d. Coliforms and Escherichia coli. The MPN technique was used to enumerate coliforms and E. coli. Brilliant Green Bile 2% broth (Oxoid) supplemented

with tryptophane and 4-methylumbelliferyl-beta-Dglucuronide (oxoid) was distributed into test tubes fitted with Durham’s tubes. The pH of the broth was 7.2 ± 0.1. The inoculated test tubes were incubated at 37°C for 24 to 48 h under aerobic conditions. In this test system, the presence of coliforms is indicated by the formation of gas bubbles in the Durham’s tubes. The coliform positive tubes are checked under ultraviolet light (366 nm). Kovaćs indole reagent (Merck) is then added to the tubes showing fluorescence under ultraviolet light, and the presence of E. coli is confirmed by the formation of a red ring after 1 to 2 min. Control cultures providing these observations were made, and the test samples were compared to the positive controls. Measurement of Acidity pH. The pH value of samples was measured at room temperature with an HI 8521 pH-Meter and combined glass electrode (Hanna Instruments Deutschland GmbH, Karlsruhe, Germany) standardized with pH 4.00 and 7.00 standard buffer solutions (Merck). Titratable acidity. Samples (20 ml) were titrated with 0.1 M NaOH, using phenolphtalein as the indicator. The amount of NaOH used (in milliliter) was multiplied by two; thus acidity was obtained in Soxhlet-Henkel degrees (Ketting, 1981). The results were expressed as percentage of lactic acid. Statistical Analysis The effect of the S. platensis biomass on the characteristic microflora, pH, and acidity of fermented ABT milks during storage was analyzed with the Student’s t-test, by means of the STATISTICA 4.5 software (StatSoft, 1994). Significance of difference was set at P < 0.05 in all cases. RESULTS AND DISCUSSION

Storage at 15°C The survival of the characteristic microflora in fermented ABT milk samples stored at 15°C is illustrated in Tables 1 and 2. Streptococcus thermophilus was the most numerous component, as its count exceeded the value of 109 cfu/ ml at the beginning of storage time. This finding is in line with the specifications given by the manufacturer for the ABT-4 starter culture (Chr. Hansen A/S, 1995). On d 0, significantly higher counts (P < 0.05) were recorded in the fermented ABT milk supplemented Journal of Dairy Science Vol. 85, No. 5, 2002

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VARGA ET AL. Table 1. Numbers1 of surviving Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium spp. in Spirulina-enriched and control fermented milks during storage at 15°C. S. thermophilus Storage time (d) 0 3 6 9 12 15 18

Spirulina 9.17 9.45 9.35 9.30 9.28 9.23 9.27

± ± ± ± ± ± ±

L. acidophilus

Control a

0.10 0.15a 0.12a 0.13a 0.11a 0.13a 0.08a

9.03 9.26 9.15 9.02 9.06 9.05 8.99

± ± ± ± ± ± ±

Spirulina b

0.06 0.18b 0.11b 0.03b 0.14b 0.11b 0.15b

7.38 7.49 7.35 7.34 7.39 7.32 7.30

± ± ± ± ± ± ±

Bifidobacterium spp.

Control a

0.07 0.07a 0.08a 0.12a 0.19a 0.11a 0.06a

7.11 7.27 7.20 7.19 7.18 7.15 7.09

± ± ± ± ± ± ±

Spirulina b

0.08 0.17b 0.14b 0.15b 0.13b 0.13b 0.16b

6.36 5.95 5.65 5.41 5.37 5.31 5.33

± ± ± ± ± ± ±

Control a

0.08 0.12a 0.09a 0.12a 0.04a 0.08a 0.14a

6.19 5.64 5.36 5.17 5.22 5.15 5.13

± ± ± ± ± ± ±

0.09b 0.15b 0.14b 0.03b 0.07b 0.03b 0.08b

Subcolumn means within row and microbial group without a common superscript differ (P < 0.05). Values are log cfu/ml means ± SD, based on nine observations (three samples, three replicates).

a,b 1

with S. platensis biomass than in controls, verifying the stimulatory effect of the cyanobacterial biomass on the growth of coccus-shaped starter bacteria (Varga et al., 1999). There was some increase in the S. thermophilus count on d 3 both in controls and in the Spirulina-supplemented product, but then a downward tendency was observed. However, the streptococcal counts were found to be within the range of 109 cfu/ml after 18 d of storage at 15°C. The difference between the numbers of streptococci in cyanobacterial samples and controls increased with storage time, reaching a value of 0.3 log cycle at the end of the storage experiment. Viability percentage of L. acidophilus during storage at 15°C was similar to that of S. thermophilus. However, it should be noted that the initial counts were 2 log cycles lower in this case than in that of streptococci. After a slight increase in viable cell counts during the first 3 d of storage, a gradual decrease was observed. Medina and Jordano (1995) found that the population of viable yogurt and probiotic organisms increased initially after manufacture, reached a maximum, and then decreased in the product during refrigerated storage. Manufacturing practices and the selection of starter cultures had the greatest effect on the survival

of S. thermophilus. Food regulations in Hungary require fermented dairy products to contain lactic acid bacteria of starter culture origin at concentrations of at least 107 cfu/g (Codex Alimentarius Hungaricus Commission, 1999). Even the counts of lactobacilli exceeded this value throughout the entire storage period. In accordance with previous reports (Chr. Hansen A/S, 1995; Medina and Jordano, 1995), the initial counts of Bifidobacterium spp. ranged between 1.0 × 106 and 5.0 × 106 cfu/ml. Loss of viability of bifidobacteria during storage was more pronounced than was that of lactic acid bacteria. Only 10% of the viability was retained after the first 9 d of storage, whereas no further significant decrease in the bifidobacterial population occurred in the course of the second 9 d. The cyanobacterial samples had 0.2 log cycle higher (P < 0.05) bifidobacterial counts than did controls at the end of storage time. The viability of bifidobacteria in fermented dairy products is a cause for concern. The survival of Bifidobacterium spp. at low pH (3.7 to 4.3) conditions was studied by Lankaputhra et al. (1996). Over 50% of the strains examined lost viability after 6 d of storage at pH 4.3. However, some strains survived well for 6 wk under all pH levels studied. High initial viable counts of bifidobacteria (> 108 cfu/

Table 2. Percent viability1 of Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium spp. in Spirulina-enriched and control fermented milks during storage at 15°C. S. thermophilus

L. acidophilus


Storage time (d)

Spirulina

Control

Spirulina

Control

Spirulina

Control

0 3 6 9 12 15 18

100.00 190.55 151.36 134.90 128.82 114.82 125.89

100.00 169.82 131.83 97.72 107.15 104.71 91.20

100.00 128.82 93.33 91.20 102.33 87.10 83.18

100.00 144.54 123.03 120.23 117.49 109.65 95.50

100.00 38.90 19.50 11.22 10.23 8.91 9.33

100.00 28.18 14.79 9.55 10.72 9.12 8.71

1 Values are % means calculated from viable count (log cfu/ml) means based on nine observations (three samples, three replicates).

Journal of Dairy Science Vol. 85, No. 5, 2002

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SPIRULINA-ENRICHED FERMENTED ABT MILK Table 3. Acidity1 of Spirulina-enriched and control probiotic fermented milks during storage at 15°C. TA2

pH Storage time (d)

Spirulina

0 3 6 9 12 15 18

4.33 4.20 4.13 4.07 4.06 4.05 4.04

± ± ± ± ± ± ±

Control

0.01a 0.01a 0.01a 0.02b 0.01b 0.01b 0.01b

4.28 4.18 4.11 4.10 4.08 4.07 4.06

± ± ± ± ± ± ±

Spirulina 0.01b 0.01b 0.01b 0.01a 0.01a 0.01a 0.01a

0.98 1.22 1.26 1.28 1.30 1.31 1.31

± ± ± ± ± ± ±

Control

0.02a 0.01a 0.01a 0.01a 0.01a 0.01a 0.02a

0.96 1.17 1.23 1.25 1.26 1.26 1.27

± ± ± ± ± ± ±

0.01b 0.01b 0.01b 0.01b 0.01b 0.01b 0.01b

Subcolumn means within row and acidity parameter without a common superscript differ (P < 0.05). Values are means ± SD, based on nine observations (three samples, three replicates). 2 Titratable acidity expressed as percentage of lactic acid. a,b 1

g) provided improved protection against acid injury to the organism. Fermented milks are characterized by low levels of oxygen, high acidity, and production of antimicrobial compounds by the starter bacteria. Therefore, some pathogenic or spoilage microorganisms cannot survive in these products (Northolt, 1983). No growth of yeasts, molds, coliform organisms, or E. coli was detected either in the cyanobacterial fermented ABT milk or in controls during the entire storage period, even though the products were manufactured under laboratory conditions and samples were stored at a relatively high temperature. Table 3 shows the changes in pH and acidity of fermented ABT products during storage at 15°C. The pH of fermented milks may decrease considerably during storage, which can affect the growth and viability of L. acidophilus and Bifidobacterium spp., most notably of the latter organism (Laroia and Martin, 1991; Hekmat and McMahon, 1992), as the growth of bifidobacteria is significantly retarded below pH 5.0 (Scardovi, 1986). It is for this reason that there was a more notable fall in the counts of bifidobacteria than in those of the other two starter-culture organisms. The pH of cyanobacterial samples was significantly

higher (P < 0.05) than that of controls at the beginning of storage time. This can be accounted for by the fact that the S. platensis biomass is of alkaline character and, although it is capable of increasing the acid production and growth of dairy starter cultures during fermentation (Varga et al., 1999), this time it was added to the product only during stirring at pH 4.5 to 4.6. However, postacidification occurred because of the elevated storage temperature and because the cyanobacterial biomass had a stimulatory effect on acid development. Approximately 0.3% lactic and acetic acids were formed during 18 d of storage at 15°C, significantly more (P < 0.05) in the cyanobacterial samples than in controls. Thus, the pH of the Spirulina-supplemented product was found to be lower (P < 0.05) than that of the control fermented milk at the end of storage time. Storage at 4°C Tables 4 and 5 illustrate the survival of characteristic microorganisms in fermented ABT milks during storage at 4°C. There was virtually no decrease in viable numbers of S. thermophilus during the first 4 wk of storage at

Table 4. Numbers1 of surviving Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium spp. in Spirulina enriched and control fermented milks during storage at 4°C. S. thermophilus Storage time (d)

Spirulina

0 7 14 21 28 35 42

9.17 9.28 9.21 9.21 9.11 9.05 8.86

± ± ± ± ± ± ±

0.10a 0.06a 0.12a 0.12a 0.07a 0.18a 0.16a

L. acidophilus

Control 9.03 9.12 9.13 9.11 9.01 8.85 8.64

± ± ± ± ± ± ±

Spirulina 0.06b 0.16b 0.08a 0.03b 0.06b 0.14b 0.18b

7.38 7.48 7.42 7.40 7.38 7.35 7.31

± ± ± ± ± ± ±

0.07a 0.08a 0.10a 0.11a 0.15a 0.07a 0.13a


Control 7.11 7.25 7.28 7.25 7.20 7.01 7.00

± ± ± ± ± ± ±

Spirulina 0.08b 0.11b 0.11b 0.13b 0.09b 0.14b 0.17b

6.36 6.42 6.34 5.88 5.91 5.23 4.86

± ± ± ± ± ± ±

0.08a 0.06a 0.03a 0.05a 0.09a 0.09a 0.05a

Control 6.19 6.30 6.17 5.82 5.73 5.07 4.69

± ± ± ± ± ± ±

0.09b 0.06b 0.08b 0.06b 0.10b 0.03b 0.08b

Subcolumn means within row and microbial group without a common superscript differ (P < 0.05). Values are log cfu/ml means ± SD, based on nine observations (three samples, three replicates).

a,b 1


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VARGA ET AL. Table 5. Percent viability1 of Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium spp. in Spirulina enriched and control fermented milks during storage at 4°C. S. thermophilus

L. acidophilus


Storage time (d)

Spirulina

Control

Spirulina

Control

Spirulina

Control

0 7 14 21 28 35 42

100.00 128.82 109.65 109.65 87.10 75.86 49.98

100.00 123.03 125.89 120.23 95.50 66.07 40.74

100.00 125.89 109.65 104.71 100.00 93.33 85.11

100.00 138.04 147.91 138.04 123.03 79.43 77.62

100.00 114.82 95.50 33.11 35.48 7.41 3.16

100.00 128.82 95.50 42.66 34.67 7.59 3.16

1 Values are % means calculated from viable count (log cfu/ml) means, based on nine observations (three samples, three replicates).

4°C, but a significant (P < 0.05) drop in survival rates of streptococci was observed afterward, with cyanobacterial samples being affected to a lesser degree than controls. The Spirulina-supplemented product contained 0.2 log cycle higher (P < 0.05) S. thermophilus counts than did the control fermented milk after 6 wk of refrigerated storage. It is of note, however, that even controls had a very high streptococcal population (> 108 cfu/ml) at this time. Our findings agreed with those of Medina and Jordano (1995), who determined that after 24 d of storage at 7°C, numbers of S. thermophilus decreased only by 11.4 to 16.8%. Robinson (1987) reported on the survival of L. acidophilus in acidophilus yogurt. The product examined gave values of 9.5 × 106, 7.6 × 106, and 4.0 × 106 cfu/ ml in an initial trial and after 7 and 14 d under refrigerated storage, respectively. In our study, similarly to what was experienced with S. thermophilus, no loss of viability of lactobacilli occurred during the first 28 d of refrigerated storage. A decrease of 0.2 log cycle in L. acidophilus count was observed in controls afterward (between d 28 and 42), whereas a higher percentage of viability of lactobacilli was retained in the cyanobacterial product during the same period. The L. acidophilus counts reached the required value of 107

cfu/ml at each sampling time, thus providing potential health benefits until 42 d of refrigerated storage. On the whole, the Spirulina-supplemented fermented ABT milk contained significantly (P < 0.05) higher levels of lactobacilli than did the control product. Reuter (1989) stated that, with adequate acidity, free from the influence of oxygen and at refrigeration temperatures, there should be no problems for bifidobacteria to remain viable even at 4 wk. In our experiment, over 95% of the viability of bifidobacteria was retained after 14 d of storage at 4°C. However, a decrease of 1.5 log cycles in the bifidobacterial population was observed during the following 4 wk, which was especially pronounced between d 28 and 35. There was no difference between the cyanobacterial samples and controls in this respect; thus, the Spirulina-supplemented fermented ABT milk contained significantly (P < 0.05) higher levels of viable bifidobacteria throughout the whole storage period than did the control product. The importance of applying a low storage temperature is clearly evidenced by the fact that the viability of bifidobacteria after 28 d of storage at 4°C was similar to that obtained after 3 d at 15°C. No growth of yeasts, molds, coliforms, or E. coli was detected in any sample, so the potential antimicrobial

Table 6. Acidity1 of Spirulina enriched and control probiotic fermented milks during storage at 4°C. TA2

pH Storage time (d) 0 7 14 21 28 35 42

Spirulina 4.33 4.30 4.27 4.23 4.24 4.25 4.24

± ± ± ± ± ± ±

Control a

0.01 0.01a 0.01a 0.01a 0.02a 0.01a 0.01a

4.28 4.28 4.26 4.24 4.25 4.26 4.23

± ± ± ± ± ± ±

Spirulina b

0.01 0.02b 0.01a 0.01a 0.01a 0.01a 0.01a

0.98 1.13 1.17 1.19 1.19 1.20 1.21

± ± ± ± ± ± ±

Control a

0.02 0.03a 0.01a 0.01a 0.01a 0.01a 0.01a

0.96 1.11 1.14 1.13 1.17 1.18 1.19

± ± ± ± ± ± ±

0.01b 0.05a 0.01b 0.01b 0.01b 0.01b 0.01b

Subcolumn means within row and acidity parameter without a common superscript differ (P < 0.05). Values are means ± SD, based on nine observations (three samples, three replicates). 2 Titratable acidity expressed as percentage of lactic acid. a,b 1


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properties of the S. platensis biomass (Falch et al., 1995; Varga and Szigeti, 1998) could not manifest themselves. Table 6 shows the changes in acidity and pH of fermented ABT milks during refrigerated storage at 4°C. No postacidification occurred at this temperature. There was a fall in pH of less than 0.1 unit in the cyanobacterial product during 42 d of storage, and the pH of controls decreased to an even lesser degree. Any increase in storage temperature seems to be of practical importance because Medina and Jordano (1995) observed a pH decline of 0.3 to 0.4 units in the case of ABT-type fermented dairy products during 36 d of storage at 7°C. In our experiment, the amount of lactic and acetic acids produced after 42 d of storage at 4°C was comparable to that measured after 3 d at 15°C. CONCLUSIONS The results of this research demonstrated that the S. platensis biomass positively influenced the survival of ABT starter bacteria regardless of storage temperature. The presence of L. acidophilus gave values within the range of 107 cfu/ml at each sampling time, and the S. thermophilus counts also exceeded the critical level by far, reaching values higher than 109 cfu/ml in most of the cases. As one would expect, bifidobacteria were highly susceptible to acid injury. Their counts fell more sharply than did those of lactobacilli and streptococci; however, the addition of cyanobacterial biomass was of beneficial effect on their viability. No spoilage organisms were detected at any sampling time, indicating the high degree of sanitation during processing and packaging of products. The storage temperature of 15°C resulted in some postacidification, whereas pH was stable during refrigerated storage at 4°C. In addition to the above benefits, the cyanobacterial biomass increased the essential amino acid and vitamin contents of cow’s milk and also improved its fatty acid composition. The abundance of bioactive components in S. platensis is of great importance from a nutritional point of view because thus the cyanobacterial biomass provides a new opportunity for the manufacture of functional dairy foods. ACKNOWLEDGMENTS This work was funded by a grant (FKFP-0197/2001) from the Ministry of Education, Hungary. The authors are grateful to G. Bogna´r (Hungarian Dairy Research Institute, Inc. at Mosonmagyaro´va´r, Hungary) and O. Pulz (Institute of Cereal Processing, Inc. at Bergholz-Rehbru¨cke, Germany) for the gener-

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