Production of probiotic Bulgarian yoghurts obtained from an ultrafiltered cow’s milk

S. Kodinova
  • Department of Process Engineering, Technical Faculty, University of Food Technologies, Plovdiv, Bulgaria
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, M. Dushkova
  • Corresponding author
  • Department of Process Engineering, Technical Faculty, University of Food Technologies, Plovdiv, Bulgaria
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, M. Miteva-Petrova
  • Department of Organic Chemical Technologies, Technical Sciences Faculty, University “Prof. Asen Zlatarov”, Burgas, Bulgaria
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, V. Yanakieva
  • Department of Microbiology, Technological Faculty, University of Food Technologies, Plovdiv, Bulgaria
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, S. Petrov
  • Department of Organic Chemical Technologies, Technical Sciences Faculty, University “Prof. Asen Zlatarov”, Burgas, Bulgaria
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and Z. Denkova
  • Department of Microbiology, Technological Faculty, University of Food Technologies, Plovdiv, Bulgaria
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Abstract

Ultrafiltration of skim cow’s milk with a UF10-PAN membrane at volume reduction ratios (VRRs) of 2 and 3 was performed. The ultrafiltration retentates obtained were used for production of probiotic yoghurts with three different starters. A control sample was prepared using skim cow’s milk. All yoghurts were analysed according to the following parameters: titratable acidity, dry matter, organoleptic characteristics, number of specific microorganisms (Lactobacillus bulgaricus and Streptococcus thermophilus) and the total count of viable lactic acid bacteria for 28 d of storage. The results showed that the increase in the VRR during ultrafiltration increased the titratable acidity, as well as the dry matter of all yoghurts. Ultrafiltration concentration led to an increase in the count of viable lactic acid bacteria in all yoghurts which improved their functional properties. The highest values of the total number of viable lactic acid bacteria were determined in yoghurts obtained with starter 1CM, followed by starters MZ2 and ZD for both VRRs. Probiotic yoghurts with the highest organoleptic evaluation were obtained from ultrafiltration retentates at VRR = 2 and starters 1CM and MZ2.

Introduction

Ultrafiltration is widely used in the dairy industry for concentration, purification and fractionation of milk components as it has the following advantages in comparison with the traditional separation methods: environmental friendliness (Kumar et al., 2013; Tamime, 2013), lower energy consumption (Baldasso et al., 2011), increased yield (Macedo et al., 2012; Ong et al., 2013) and improved quality (Reschke da Cunha et al., 2006; Domagala and Wszolek, 2008; Heino et al., 2010; Domagala et al., 2012) of the final product, reduction in the production costs (Mehaia, 2005) and completion of the process at room temperature to treat heat-sensitive products and keep their natural properties (in comparison with thermal evaporation, for example; Baldasso et al., 2011).

Many fermented milk products are produced by ultrafiltration. When ultrafiltration was used for Greek yoghurt, it was established that yoghurts produced by ultrafiltration contained more lactic acid bacteria than those produced by traditional technology without ultrafiltration (Tamime et al., 2005). Sodini et al. (2005) used whey protein concentrates, obtained by ultrafiltration, to increase the protein content and enhance the development of lactic acid bacteria in the probiotic yoghurts produced.

Ymer is a national Danish soured milk product with an increased protein content, which can be obtained by traditional technology or by using membrane processes (Fonden et al., 2006). The ultrafiltration method for obtaining Ymer includes the following processing operations: heat treatment of skim milk at 85°C for 15 s, ultrafiltration at a volume reduction ratio (VRR) of 1.9 and a temperature of 50–55°C, standardisation of the retentate by the addition of cream, reheating to 85°C for 5 min, homogenising and cooling to 22°C. The coagulation is performed by mesophilic starter culture at 20–22°C for 14–16 h. The advantage of this technology is higher protein content, which leads to an increase in yield from 8% to 15%.

Özer (2006) developed a technology with ultrafiltration for the traditional sweet strained Indian yoghurt Shrikhand: skim cow’s milk was subjected to pasteurisation at 85–90°C for 10–20 min; then, it was cooled at 21–22°C and coagulated with mesophilic lactic acid bacteria during 15–16 h. The fermented milk was reheated to 60°C for 5 min, cooled at 50°C and subjected to ultrafiltration to increase the dry matter to 16%. The product obtained had a better taste, texture, colour and appearance than that obtained by traditional technology.

Kumis is an ancient fermented milk drink, commonly consumed in Eastern Europe and Central Asia. Traditionally, it is made from mare’s milk, and its healing and nutritional properties are well known but the quantity of mare's milk is limited and the price is quite high. Küçükçetin et al. (2003) investigated the possibility of using cow’s milk for the production of Kumis: skim cow’s milk was treated by ultrafiltration to obtain a protein-enriched concentrate. The casein and whey proteins in the ultrafiltration concentrate were separated by microfiltration, and the resulting retentates had a composition close to the mare’s milk.

Labneh is a traditional fermented milk product, popular in different parts of the world, especially in the Balkans. It has a sour taste, milky white colour, smooth and creamy texture. In traditional Labneh technology, whole yoghurt is drained through filtering tissue to obtain dry matter from 22% to 26% (Otaibi and Demerdash, 2008). A comparative assessment of the chemical composition, rheological and organoleptic properties of Labneh obtained from cow’s milk using traditional technology and using ultrafiltration retentate with or without added concentrated permeate was made (Shamsia and El-Ghannam, 2012). The authors found that the addition of 1% concentrated permeate containing 84% lactose, 11% mineral substances, 5% water and 1% glucono delta-lactone (GDL) resulted in a significant reduction in coagulation time and an increase in dry matter. The most significant reduction in coagulation time was observed when using a GDL. Compared with Labneh, obtained by traditional technology, when ultrafiltration was used the product was characterised by a higher content of total and soluble proteins, fats, minerals, acidity and pH. The addition of 1% concentrated permeate during the production of Labneh by ultrafiltration results in an improvement in taste, appearance and structure of the product.

Mehaia (2005) explored the possibility of the production of Labneh from goat’s milk by traditional technology and by using ultrafiltration before and after coagulation with starter culture. The author found that Labneh, produced by membrane technology, had higher acidity, higher protein, fat, dry matter and lower pH. Ultrafiltration before and after coagulation led to an increase in yield of about 14.5%. The production time was significantly reduced by 75%, as well as the amount of starter used before (12.5%) and after (62.5%) ultrafiltration.

The aim of this research was to investigate the possibilities for the production of probiotic Bulgarian yoghurts obtained by ultrafiltration of skim cow’s milk with a UF10-PAN membrane and assessment of their physicochemical, microbiological and organoleptic characteristics.

Materials and methods

Materials

Milk

The skim cow’s milk was delivered by BCC Handel Ltd., Elena, Bulgaria. The milk was analysed for the following parameters: dry matter content (International Standardisation Organisation [ISO], Geneva, Switzerland 6731:2010); total protein content (Bulgarian State Standard [BSS] EN ISO 8961-1:2014); fat content (ISO 2446:2008); mineral substances (BSS 6154:1974). All these analyses were conducted with threefold repetition.

Starter cultures

Three probiotic starter cultures were used for the production of Bulgarian yoghurts: starter culture ZD consisting of a probiotic strain of Lactobacillus bulgaricus (National Bank for industrial microorganisms and cell cultures NBIMCC 3706) and Streptococcus thermophilus (3); starter culture MZ2 consisting of a probiotic strain of L. delbrueckii subsp. bulgaricus (NBIMCC 3708) and S. thermophilus (TMZ2 I); starter culture 1CM consisting of a probiotic strain of L. delbrueckii subsp. bulgaricus (NBIMCC 3708) and S. thermophilus (T3).

The ratio of L. delbrueckii subsp. bulgaricus and S. thermophilus was 1:2 in all starter cultures. The starter cultures were kindly provided by Prof. Zapryana Denkova from the Department of Microbiology at University of Food Technologies, Plovdiv, Bulgaria.

Media for development and maintenance of lactic acid bacteria: Sterile skim milk with a titratable acidity of 16–18°T – dried skim milk was provided by Scharlau, Barcelona, Spain, reconstituted to 9% dry matter content, autoclaved for 15 min at 118°C and cooled for storage at room temperature. Liquid medium (LAPTg10) for the development of lactic acid bacteria was prepared as follows: peptone – 15.0 kg/m3 (Fluka, Bucharest, Romania); tryptone – 10.0 kg/m3 (Fisher Scientific, Difco Laboratories, Hampton, USA); yeast extract – 10.0 kg/m3 (Scharlau), glucose – 10.0 kg/m3 (Sigma Aldrich, Merck, St. Louis, MO, USA); Tween 80 – 1.0 kg/m3 (Sigma Aldrich). The pH of the liquid medium was 6.6–6.8 and the solid medium of LAPTg10 was 15.0 kg/m3 agar (Sigma Aldrich).

Methods

Cultivation and storage of probiotic starter cultures for yoghurt The starter cultures used (ZD, MZ2, 1CM) were inoculated every 20 d in sterile skim milk with a titratable acidity of 16–18°T and stored at 4–6°C or as stock cultures at −20°C.

Ultrafiltration experiments

Ultrafiltration was carried out with polyacrylonitrile membrane UF10-PAN with 10 kDa molecular weight cut-off. Membrane was prepared by the dry–wet phase inversion method of polymer solutions with a solvent of dimethyl sulphoxide (Sigma Aldrich). Then, it was heat-treated in an aqueous medium for 10 min at 60°C. The membrane was prepared and kindly provided by the University Prof. Dr. Asen Zlatarov, Burgas, Bulgaria. Ultrafiltration experiments were carried out on laboratory equipment with a replaceable plate and frame membrane module (Figure 1). Ultrafiltration was undertaken at the following operating conditions: VRR = 2 and VRR = 3; working pressure, 0.5 MPa; temperature, 50°C; volumetric flow rate, 330 dm3/h. The retentates obtained were then pasteurised at 65°C during 10–15 min and cooled at 42 ± 1°C. VRR was calculated by the following formula:

VRR=V0VR
where V0 is the volume of the feed solution and VR is the volume of retentate.

Figure 1
Figure 1

Scheme of laboratory equipment with a replaceable plate and frame membrane module 1: valve; 2, 3, 4: manometers; 5: replaceable plate and frame membrane module; 6: pump; 7: tank for initial solution; 8: cylinder for permeate.

Citation: Irish Journal of Agricultural and Food Research 59, 1; 10.2478/ijafr-2020-0001

Production of probiotic Bulgarian yoghurts

The coagulation of cow’s milk and retentates was performed under aseptic conditions in sterile plastic containers of 100 cm3 with 1.5% probiotic starter. The containers were placed in an incubator at 41–42°C for the coagulation of milk or retentates for 2.5–3 h. After coagulation, the yoghurts were cooled and stored at 2–6°C for 28 d.

Analysis of milk, retentates and yoghurts

The initial skim cow’s milk and retentates obtained were analysed according to titratable and active acidity, total number of mesophilic anaerobic and facultative anaerobic microorganisms, as well as specific microorganisms, while the yoghurts were analysed according to dry matter, protein content, titratable acidity, specific microorganisms, number of viable lactic acid bacteria and organoleptic characteristics using the following methods.

Physicochemical methods

Dry matter was measured according to ISO 6731:2010; total protein content was investigated according to BSS EN ISO 8961-1:2014. The ability of lactic acid bacteria to form acids (titratable acidity, °T) was measured by the Turner method according to BSS 1111:1980. 1°T was equal to 1 cm3 of 0.1 N NaOH (Sigma Aldrich), necessary for neutralisation of an equivalent quantity of organic acid in 100 cm3 of culture medium. 10 cm3 from every sample was taken, and 20-cm3 distilled water was added. The titration was performed with 0.1 N NaOH using an indicator phenolphthalein until the appearance of light pink coloration, persistent for 1 min. To measure the active acidity (pH) a pen-type pH metre (PH-03 [I]; Hinotek, China) was used. All these analyses were conducted with threefold repetition.

Microbiological methods

The number of viable lactic acid bacteria was measured as appropriate serial dilutions of the yoghurts in saline solution NaCl (5 g/dm3; Sigma Aldrich) were prepared and the spread plate method was applied. 0.1 cm3 of the last three dilutions was used to inoculate in LAPTg10-agar for 3 d at 37°C until the appearance of countable single colonies. The total number of mesophilic anaerobic and facultative anaerobic microorganisms was measured according to BSS EN ISO 4833-1:2013. The count of Escherichia coli was established according to BSS EN ISO 16649-2:2014. The number of Staphylococcus aureus was identified according to BSS EN ISO 6888-1:2005+A1:2005. The concentration of Salmonella was defined according to BSS EN ISO 6579:2003. To measure yeasts and moulds, BSS EN ISO 6611:2006 was used. All these analyses were conducted with threefold repetition.

Organoleptic analysis

Organoleptic analysis was performed using a 5-point hedonic scale for evaluation, and the basic organoleptic indices are presented in Table 1. A nine-member experienced panel drawn from the Department of Microbiology at the University of Food Technologies, Plovdiv, Bulgaria, was used to evaluate the samples. The panellists rated the samples three times in a random order for colour, appearance of coagulum, structure at cutting, consistency at shattering, taste and aroma. Room temperature water and unsalted crackers were given to the panellists for mouth rinsing between samples to eliminate carry-over effects.

Table 1

Organoleptic analyses of indices and hedonic scale for evaluation of probiotic Bulgarian yoghurts

Organoleptic indices for evaluation of probiotic Bulgarian yoghurts
IndicesCharacteristics and norms
1. ColourWhite with different shades of creamy hue depending on the raw materials used
2. Appearance of coagulumDense, smooth, lateral tear is allowed depending on the type of milk
3. Structure at cuttingSmooth surface, with or without a grain-shaped structure, with or without a slight separation of the wheydepending on the raw materials used
4. Consistency at shatteringUniform, homogeneous, cream-like, light-grained or grained structure depending on the raw material used
5. Taste and aromaA pleasant, lactic acid. No side taste and odour is allowed
Hedonic scale for evaluation of probiotic Bulgarian yoghurts
EvaluationPoints
I dislike extremely1
I dislike2
I neither like nor dislike3
I like4
I like extremely5

Statistical method

The least significant difference (LSD) method was used at the level of significance 0.05, using Microsoft Excel 2010, for comparison between the control and retentates VRR = 2 and VRR = 3, as well as between the three starter cultures.

Results

The main components, titratable acidity and pH of the initial skim milk and ultrafiltration retentates at VRR = 2 and VRR = 3 are presented in Table 2. It can be seen that the increase in VRR led to an increase in the dry matter, protein, fat contents and mineral substances. The experimental results of the titratable acidity and pH showed that the lowest values of the titratable acidity were observed for the control followed by the ultrafiltration retentates at VRR = 2 and 3. Titratable acidity increased from 23 ± 0.36°T (VRR = 2) to 31 ± 0.09°T (VRR = 3) in comparison with the control (16 ± 0.18°T). Table 2 also shows that the pH decreased when using the ultrafiltration process.

Table 2

Main components and chemical properties of initial skim milk and ultrafiltration retentates at VRR = 2 and VRR = 3

IndicesSampleAverage values ± s.d.

123

Membrane UF10-PAN
Skim milk
Dry matter content, %8.908.858.878.87 ± 0.03a
Total protein content, %3.213.263.273.25 ± 0.03a
Fat content, %0.10.10.050.08 ±0.04a
Mineral substances, %0.710.720.720.72 ± 0.01a
Titratable acidity, °T15.8216.016.1816.0 ±0.18a
pH6.756.766.746.75 ± 0.01a
VRR = 2
Dry matter content, %12.2312.2412.2512.24 ± 0.01b
Total protein content, %5.595.595.745.64 ± 0.09b
Fat content, %0.20.20.10.17 ± 0.06a
Mineral substances, %0.960.960.970.96 ± 0.01b
Titratable acidity, °T22.642323.3623 ± 0.36b
pH6.606.656.636.62 ± 0.02b
VRR = 3
Dry matter content, %15.3315.3715.3515.35 ± 0.02c
Total protein content, %6.937.377.397.23 ± 0.26c
Fat content, %0.30.30.150.25 ± 0.09a
Mineral substances, %1.241.241.251.24 ± 0.01c
Titratable acidity, °T30.913131.0931 ± 0.09c
pH6.506.526.516.51 ± 0.01c
a–c

To compare the composition of skim milk and retentates at VRR = 2 and VRR = 3.

VRR, volume reduction ratio.

The results of the total number of mesophilic aerobic and facultative anaerobic microorganisms, specific microorganisms (E. coli, S. aureus and Salmonella, moulds and yeasts in the initial skim milk and ultrafiltration retentates show that the increase in VRR led to an increase in the total number of mesophilic aerobic and facultative anaerobic microorganisms (P < 0.05). The lowest values were found for the control (1.8 x 102 ± 0.1 x 102 cfu/cm3), followed by the ultrafiltration retentate at VRR = 2 (2.5 x 102 ± 0.1 x 102 cfu/cm3) and VRR = 3 (3.8 x 102 ± 0.13 x 102 cfu/cm3). The analysis for specific microorganisms in probiotic yoghurts obtained from the initial skim milk (control) and ultrafiltration retentates at VRRs of 2 and 3 showed that E. coli and S. aureus were less than 10 cfu/g, and Salmonella was not found in 25 g of the product. The count of moulds and yeasts was below 10 cfu/g in all tested probiotic yoghurts.

The results of the dry matter and protein content of the probiotic yoghurts obtained are presented in Table 3. The dry matter content of the controls was as follows: for ZD, (8.80 ± 0.14%); for MZ2, (8.87 ± 0.16%); for 1CM, (8.90 ± 0.11%). The dry matter of the yoghurts obtained from ultrafiltration retentate at VRR = 2 was as follows: for ZD, (12.20 ± 0.10%); for MZ2, (12.25 ± 0.12%); for 1CM, (12.40 ± 0.10%). The highest values were defined at VRR = 3: for ZD, (15.30 ± 0.16%); for MZ2, (15.35 ± 0.12%); for 1CM, (15.38 ± 0.13%). The data show that the highest values of the protein content were defined for yoghurts obtained from retentate at VRR = 3: for ZD, (7.23 ± 0.04%); for MZ2, (7.30 ± 0.05%); for 1CM, (7.34 ± 0.05%).

Table 3

Dry matter and protein content of probiotic Bulgarian yoghurts from initial skim milk (control) and ultrafiltration retentates at VRR = 2 and VRR = 3

Probiotic yoghurts with different startersDry matter, %Average values ± s.d.Protein content, %Average values ± s.d.


123123
ZD (control)8.668.808.948.80 ± 0.14a3.223.243.283.25 ± 0.03a
ZD (VRR = 2)12.1012.2012.3012.20 ± 0.10b5.605.705.635.64 ± 0.05b
ZD (VRR = 3)15.1415.3015.4615.30 ± 0.16c7.287.207.227.23 ± 0.04c
MZ2 (control)8.718.879.038.87 ± 0.16a3.263.303.313.29 ± 0.03a
MZ2 (VRR = 2)12.1312.2512.3712.25 ± 0.12b5.705.745.625.69 ± 0.06b
MZ2 (VRR = 3)15.2315.3515.4715.35 ± 0.12c7.257.317.357.30 ± 0.05c
1CM (control)8.798.909.018.90 ± 0.11a3.323.283.323.31 ± 0.02a
1CM (VRR = 2)12.3012.4012.5012.40 ± 0.10b5.815.695.665.72 ± 0.08b
1CM (VRR = 3)15.2515.3815.5115.38 ± 0.13c7.307.337.407.34 ± 0.05c
a–c

To compare the dry matter and protein content of the obtained yoghurts with three probiotic starters (ZD, MZ2, 1CM), and they indicate that mean values in the columns are significantly different (P < 0.05).

VRR, volume reduction ratio.

The changes in the number of Lactobacillus bulgaricus, S. thermophilus, as well as the total number of viable lactic acid bacteria for 28-day storage at a temperature of 2–6°C for all probiotic yoghurts were investigated. The results of experimental investigations are shown in Figures 24. The comparison of L. bulgaricus for each of the storage stages for the three types of yoghurt (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter ZD (Figure 2) showed that on the first day of the storage period the number of rod-shaped forms was higher (P < 0.05) at VRR = 2 (2 x 1010 ± 0.35 x 1010 cfu/g) and VRR = 3 (3 x 1010 ± 0.5 x 1010 cfu/g) in comparison with the control – 1 x 1010 ± 0.35 x 1010 cfu/g. Similar results were obtained for coccus-shaped forms – 3 x 1010 ± 0.35 x 1010 cfu/g at VRR = 2 and 3.2 x 1010 ± 0.5 x 1010 cfu/g at VRR = 3 in comparison with 1.7 x 1010 ± 0.35 x 1010 cfu/g in the control. The total count of viable lactic acid bacteria was highest in yoghurt obtained from ultrafiltration retentate at VRR = 3 (6.2 x 1010 ± 0.5 x 1010 cfu/g), followed by ultrafiltration retentate at VRR = 2 (5 x 1010 ± 0.35 x 1010 cfu/g) and control (2.7 x 1010 ± 0.35 x 1010 cfu/g). The concentration of viable cells of the probiotic strain L. bulgaricus, S. thermophilus and the total number of viable lactic acid bacteria remained high during the whole storage period, above – 2 x 108 cfu/g, as the strongest reduction was observed on the 28th day of the storage period.

Figure 2
Figure 2

Microbiological status of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter ZD. VRR, volume reduction ratio.

Citation: Irish Journal of Agricultural and Food Research 59, 1; 10.2478/ijafr-2020-0001

Figure 3
Figure 3

Microbiological status of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter MZ2. VRR, volume reduction ratio.

Citation: Irish Journal of Agricultural and Food Research 59, 1; 10.2478/ijafr-2020-0001

Figure 4
Figure 4

Microbiological status of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter 1CM. VRR, volume reduction ratio.

Citation: Irish Journal of Agricultural and Food Research 59, 1; 10.2478/ijafr-2020-0001

The change in L. bulgaricus and S. thermophilus, as well as the total number of lactic acid bacteria of probiotic yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter MZ2, is presented in Figure 3. The results indicate that on the first day of the storage period the number of rod-shaped forms was higher (P < 0.05) at VRR = 2 (2.8 x 1010±0.34 x 1010 cfu/g) and VRR = 3 (3.8 x 1010 ± 0.20 x 1010 cfu/g) in comparison with the control – 2 x 1010 ± 0.32 x 1010 cfu/g. A similar trend was observed for the coccus-shaped forms: 1.1 x 1010 ± 0.32 x 1010 cfu/g in control in comparison with 1.5 x 1010 ± 0.34 x 1010 cfu/g at VRR = 2 and 4 x 1010 ± 0.20 x 1010 cfu/g at VRR = 3. The amount of viable cells was kept high during all storage periods, and on the 28th day it was above 8 x 108 cfu/g.

The dynamics of the change in L. bulgaricus, S. thermophilus and the total number of lactic acid bacteria of probiotic yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter 1CM is presented in Figure 4. The data show that their concentration is greatest in yoghurt from ultrafiltration retentate at VRR = 3, followed by the yoghurt from ultrafiltration retentate at VRR = 2 and the control. A reduction in the amount of viable lactic acid bacteria was observed during the studied storage period, and the biggest decrease was observed from the 21st to the 28th day.

The titratable acidity of the yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with three types of probiotic starters was determined (Figure 5). The results show that the titratable acidity of all yoghurts increased (P < 0.05) with VRR of the milk used in manufacture and with storage time.

Figure 5
Figure 5

Kinetics of titratable acidity of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starters ZD, MZ2 and 1CM. VRR, volume reduction ratio.

Citation: Irish Journal of Agricultural and Food Research 59, 1; 10.2478/ijafr-2020-0001

The results of organoleptic evaluation of the probiotic yoghurts are presented in Table 4. The data show that the yoghurts from retentate at VRR = 2 with all starter cultures had the highest number of points. Yoghurts, which had the higher total number of points, were these with starters MZ2 and 1CM in comparison with starter ZD.

Table 4

Organoleptic characteristics of probiotic Bulgarian yoghurts from skim milk (control and retentates at VRR = 2 and VRR = 3) with different starters

IndicesType of probiotic yoghurt
Starter ZD
ControlVRR = 2VRR = 3
Appearance of coagulumLoose, smooth coagulum with slight lateral tearing during inclination of the package – 4 pointsDense, smooth coagulum – 4 pointsDense, grainy coagulum – 4 points
Consistency at shatteringHomogenous – 4 pointsHomogenous – 5 pointsHomogenous – 4 points
ColourWhite with creamy hue – 5 pointsWhite with creamy hue – 5 pointsWhite with creamy hue – 5 points
Structure at cuttingSmooth surface, with abundant separation of whey – 3 pointsSmooth surface, with slight separation of whey – 4 pointsSmooth surface, with slight separation of whey – 4 points
Taste and aromaSlight cream-like taste – 2 pointsSlight cream-like taste – 4 pointsSlight cream-like taste – 3 points
Total points18 points22 points20 points
Starter MZ2
ControlVRR = 2VRR = 3
Appearance of coagulumLoose, smooth coagulum with slight lateral tearing during inclination of the package – 4 pointsDense, smooth coagulum – 5 pointsDense coagulum – 3 points
Consistency at shatteringHomogenous – 5 pointsHomogenous – 5 pointsHomogenous – 4 points
ColourWhite with creamy hue – 5 pointsWhite with creamy hue – 5 pointsWhite with creamy hue – 5 points
Structure at cuttingSmooth surface, with abundant separation of whey – 3 pointsSmooth surface, with slight separation of whey – 4 pointsSmooth surface, with slight separation of whey – 4 points
Taste and aromaSlight cream-like taste – 5 pointsPleasant cream-like taste – 4 pointsStrong cream-like taste – 3 points
Total points22 points23 points19 points
Starter 1CM
ControlVRR = 2VRR = 3
Appearance of coagulumLoose, smooth coagulum with slight lateral tearing during inclination of the package – 3 pointsDense, smooth coagulum – 4 pointsDense, grainy coagulum – 4 points
Consistency at shatteringHomogenous – 4 pointsHomogenous – 5 pointsHomogenous – 3 points
ColourWhite with creamy hue – 5 pointsWhite with creamy hue – 5 pointsWhite with creamy hue – 5 points
Structure at cuttingSmooth surface, with abundant separation of whey – 4 pointsSmooth surface, with slight separation of whey – 5 pointsSmooth surface, with slight separation of whey – 5 points
Taste and aromaSlight cream-like taste – 4 pointsPleasant cream-like taste – 4 pointsStrong cream-like taste – 3 points
Total points20 points23 points20 points

VRR, volume reduction ratio.

Discussion

Table 2 shows that the increase in VRR led to an increase in titratable acidity (P < 0.05). This could be explained by the higher protein content obtained with ultrafiltration. Moreno-Montoro et al. (2015) reported that the increased protein content leads to a higher buffering capacity which results in greater titratable acidity. The authors established significantly higher values of titratable acidity in ultrafiltered retentates from goat’s milk in comparison with skim goat’s milk and enhanced nutritional value because of the increase in dry matter, protein, fat contents and mineral substances. The increase in VRR resulted in a decrease in the active acidity (pH) of the investigated samples (P < 0.05).

Low levels of ultrafiltration concentration (VRR = 2 and VRR = 3) were used for the production of probiotic yoghurts because at higher levels the dry matter and protein content increase the density and the viscosity of the milk, which slows down the acid coagulation (Meletharayil et al., 2015; Arango et al., 2018).

The increase in the total number of mesophilic aerobic and facultative anaerobic microorganisms in retentates could be explained by the decrease in the volume of the initial skim milk and higher concentration of microorganisms during ultrafiltration. It can be seen that in microbiological analysis, the initial skim milk and ultrafiltration retentates at VRR = 2 and 3 were in agreement with the admissible hygienic and epidemiological assessment norms according to the instruction from 31 July 2004 for the Maximum Allowable Quantities of Pollutants in Foods (Official Journal of Bulgarian Government, issue 88/8, 2004).

The statistical analysis of the data in Table 3 shows that there was no significant difference (P > 0.05) between the dry matter content of the yoghurts obtained with the three probiotic starters (ZD, MZ2, 1CM) in all tested combinations. It can also be seen that the increase in VRR led to an increase in the dry matter content of the samples which could be explained by the volume reduction during ultrafiltration concentration.

There was an increase in concentration due to an increase in the total number of lactic acid bacteria (Figure 2). Similar results were reported by Damianova et al. (2009) who demonstrated that the addition of plant proteins stimulated the development and viability of lactic acid bacteria and contributed to maintaining their higher amounts in the final lactic acid product. Marafon et al. (2011) found that the addition of whey protein concentrate and sodium caseinate resulted in an increase in the number of viable cells of L. bulgaricus, S. thermophilus and the probiotic strain of Bifidobacterium animalis in the yoghurts obtained.

Figure 3 shows that the lowest values of the total number of lactic acid bacteria were observed in the controls, followed by the ultrafiltration retentates at VRR = 2 and VRR = 3. This could be explained by the different dry matter content in the samples investigated. Mahdian and Tehrani (2007) reported that increased dry matter content keeps higher concentrations of L. bulgaricus and S. thermophilus in the yoghurt obtained.

There was research on the change in viable probiotic bacteria in the production of stirred-type yoghurt from goat’s milk (Martìn-Diana et al., 2003). The authors found that the addition of 3% whey protein concentrate resulted in an increase in the amount of S. thermophilus ST-20Y, L. acidophilus LA-5 and Bifidobacterium BB-12 – from 6.4 log units to 8.7 log units due to the increased protein content. Figure 3 also shows that a reduction was observed during the storage period and the biggest decrease was from the 21st to the 2th day. Similar results were also obtained from the experimental work of Oliveira et al. (2002) on the 28th day of storage in lactic acid beverages.

Comparing the total number of viable lactic acid bacteria of the three starter cultures used, it could be seen that the highest values were observed in the yoghurt with starter 1CM (P < 0.05), followed by the starter MZ2 (P < 0.05) and starter ZD (P < 0.05). This dependence concerns both controls and yoghurts derived from ultrafiltration retentates at VRR = 2 and VRR = 3.

Figure 5 shows that the lowest values of titratable acidity were obtained in the controls, followed by yoghurts from ultrafiltration retentates at VRR = 2 and VRR = 3. This was probably due to the higher protein content in ultrafiltered retentates, which was favourable for the growth of lactic acid bacteria. This led to a higher concentration of lactic acid. The strongest increase in titratable acidity was observed in the period from 1 to 14 d, after which it remained practically unchanged. This could be explained by the inhibition in the growth of lactic acid bacteria from accumulated lactic acid during the storage period (Kondratenko and Simov, 2003). Figure 5 shows that yoghurt with the highest acidity was obtained with starter 1CM, followed by starters MZ2 and starters ZD. This was probably due to the higher content of S. thermophilus in the yoghurts examined. The pH of the yoghurts was not measured as it is likely to have decreased with VRR owing to the increase in titratable acidity.

Conclusion

The results show that the increase in VRR led to an increase in the titratable acidity of initial milk, ultrafiltered retentates and yoghurts obtained. The level of ultrafiltration concentration led to an increase in the count of viable lactic acid bacteria in all yoghurts which improved their functional properties. The highest values of the total number of viable lactic acid bacteria were determined in Bulgarian yoghurts obtained with starter 1CM, followed by starters MZ2 and ZD. Probiotic yoghurts with the highest organoleptic evaluation were obtained from ultrafiltration retentates at volume reduction ratio VRR = 2 and starters 1CM and MZ2.

References

  • Arango, O., Trujillo, A.J. and Castillo, M. 2018. Monitoring the effect of inulin, protein, and calcium on milk coagulation phases using a fibre optic sensor. International Dairy Journal 81: 80–86.

  • Baldasso, C., Barros, T.C. and Tessaro, I.C. 2011. Concentration of whey proteins by ultrafiltration. Desalination 278: 381–386.

  • Bulgarian State Standard BSS 1111:1980. Milk and milk products – determination of acidity.

  • Bulgarian State Standard BSS 6154:1974. Milk and milk products. Methods for determination of ash content.

  • Bulgarian State Standard BSS EN ISO 4833-1:2013. Microbiology of the food chain – horizontal method for the enumeration of microorganisms – part 1: Colony count at 30°C by the pour plate technique.

  • Bulgarian State Standard BSS EN ISO 6579: 2003. Microbiology of food and animal feeding stuffs – horizontal method for the detection of Salmonella spp (ISO 6579:2002).

  • Bulgarian State Standard BSS EN ISO 6888-1:2005 + A1: 2005. Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) – part 1: technique using Baird-Parker agar medium – amendment 1: inclusion of precision data (ISO 6888-1:1999/Amd 1:2003).

  • Bulgarian State Standard BSS EN ISO 16649-2:2014. Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – part 2: Colony count technique at 44°C using 5-bromo-4-chloro-3-indolul beta-D-glucuronide.

  • Bulgarian State Standard BSS ISO 6611:2006. Milk and milk products – enumeration of colony-forming units of yeasts and/or moulds – colony-count technique at 25°C.

  • Damianova, S., Vasileva, N., Todorova, S., Stefanova, R. and Ga-neva, E. 2009. Obtaining of functional food products. I. Yogurt with oat meal. Proceedings of Rouse University, Bulgaria, Volume 48, pages 169–174. (In Bulgarian).

  • Domagala, J. and Wszolek, M. 2008. Effect of concentration method and starter culture type on the texture and susceptibility to syneresis of yoghurt and bio-yoghurts made of goat‘s milk. Zywnosc-Nauka, Technologia, Jakosc 15: 118–128.

  • Domagala, J., Wszolek, M. and Dudzinska, A. 2012. The influence of the fortification method and starter culture type on the texture and microstructure of probiotic yoghurts prepared from goat‘s milk. Milchwissenschaft 67: 172–176.

  • Fonden, R., Leporanta, K. and Svensson, U. 2006. Nordic/Scandinavian fermented milk products. In: “Fermented Milks”, 1st Edition (ed. A.Y. Tamime), Blackwell Science Ltd, Oxford, UK, pages 156–173.

  • Heino, A., Uusi-Rauva, J. and Outinen, M. 2010. Pre-treatment methods of Edam cheese milk. Effect on cheese yield and quality. Journal of Food Science and Technology 43: 640–646.

  • ISO 2446:2008. Milk – determination of fat content.

  • ISO 6731:2010 (IDF 21:2010). Milk, cream and evaporated milk – determination of total solids content (Reference method).

  • ISO 8968-1:2014 (IDF 20-1:2014). Milk and milk products – determination of nitrogen content – part 1: Kjeldahl principle and crude protein calculation.

  • Kondratenko, M.S. and Simov, J.I. 2003. “Bulgarian yoghurt”. Association of Dairy Processors in Bulgaria, Sofia, 262. (In Bulgarian).

  • Küçükçetin, A., Yagin, H., Hinrichs, J. and Kulozik, U. 2003. Adaptation of bovine milk towards mare’s milk composition by means of membrane technology for koumiss manufacture. International Dairy Journal 13: 945–951.

  • Kumar, P., Sharma, N., Ranjan, R., Kumar, S., Bhat, Z. and Jeong, D. 2013. Perspective of membrane technology in dairy industry: a review. Asian-Australasian Journal of Animal Sciences 26: 1347–1358.

  • Macedo, A., Pinho, M. and Duarte, E. 2012. Application of ultrafiltration for valorization of ovine cheese whey. Procedia Engineering 44: 1949–1950.

  • Mahdian, E. and Tehrani, M. 2007. Evaluation of the effect of milk total solids on the relationship between growth and activity of starter cultures and quality of concentrated yoghurt. American-Eurasian Journal of Agricultural and Environmental Sciences 2: 587–592.

  • Marafon, A.P., Sumi, A., Alcantara, M.R., Tamime, A.Y. and Oliveira, M.N. 2011. Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins. LWT – Food Science and Technology 44: 511–519.

  • Martìn-Diana, A.B., Janer, C., Peláez, I. and Requena, T. 2003. Development of a fermented goat’s milk containing probiotic bacteria. International Dairy Journal 13: 827–833.

  • Mehaia, M.A. 2005. Manufacture of fresh Labneh from goat’s milk using ultrafiltration process. Journal of Food Technology 3: 24–29.

  • Meletharayil, G.H., Patel, H.A. and Huppertz, T. 2015. Rheological properties and microstructure of high protein acid gels prepared from reconstituted milk protein concentrate powders of different protein contents. International Dairy Journal 47: 64–71.

  • Moreno-Montoro, M., Olalla, M., Giménez-Martínez, R., Bergillos-Meca, T., Ruiz-López, M., Cabrera-Vique, C., Artacho, R. and Miguel Navarro-Alarcón, M. 2015. Ultrafiltration of skim goat’s milk increases its nutritional value by concentrating nonfat solids such as proteins, Ca, P, Mg, and Zn. Journal of Dairy Science 98: 7628–7634.

  • Oliveira, M.N., Sodini, I., Remeuf, F., Tissier, J.P. and Corrieu, G. 2002. Manufacture of fermented lactic beverages containing pro-biotic cultures. Journal of Food Science 67: 2336–2341.

  • Ong, L., Dagastine, R., Kentish, S. and Gras, S. 2013. Microstructure and composition of full fat Cheddar cheese made with ultrafiltered milk retentate. Foods 2: 310–331.

  • Otaibi, M.A. and El Demerdash, H. 2008. Improvement of the quality and shelf life of concentrated yoghurt (labneh) by the addition of some essential oils. African Journal of Microbiology Research 2: 156–161.

  • Özer, B.H. 2006. Production of concentrated products. In: “Fermented Milks”, 1st Edition (ed. A.Y. Tamime), Blackwell Science Ltd, Oxford, UK, pages 128–155.

  • Reschke da Cunha, C., Viotto, W.H. and Viotto, L.A. 2006. Use of low concentration retentates in reduced fat “Minas Frescal” cheese manufacture: effect on composition, proteolysis, viscoelastic properties and sensory acceptance. International Dairy Journal 16: 215–224.

  • Shamsia, S.M. and El-Ghannam, M.S. 2012. Manufacture of Labneh from cow’s milk using ultrafiltration retentate with or without addition of permeate concentrate. Journal of Animal Production Advances 2: 166–173.

  • Sodini, I., Montella, J. and Tong, P.S. 2005. Physical properties of yogurt fortified with various commercial whey protein concentrates. Journal of the Science of Food and Agriculture 85: 853–859.

  • Tamime, A.Y. 2013. In: “Membrane Processing: Dairy and Beverage Applications”, 1st Edition (ed. A.Y. Tamime), Development of membranes processes, Wiley-Blackwell, USA, pages 4–158.

  • Tamime, A.Y., Saarela, M., Sondergard, A.K., Mistry, V.V. and Shah, N.P. 2005. Production and maintenance of viability of probiotic microorganisms in dairy products. In: “Probiotic Dairy Products”, 1st Edition (ed. A.T. Tamime), Blackwell Science Ltd, Oxford, UK, pages 39–72.

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  • Arango, O., Trujillo, A.J. and Castillo, M. 2018. Monitoring the effect of inulin, protein, and calcium on milk coagulation phases using a fibre optic sensor. International Dairy Journal 81: 80–86.

  • Baldasso, C., Barros, T.C. and Tessaro, I.C. 2011. Concentration of whey proteins by ultrafiltration. Desalination 278: 381–386.

  • Bulgarian State Standard BSS 1111:1980. Milk and milk products – determination of acidity.

  • Bulgarian State Standard BSS 6154:1974. Milk and milk products. Methods for determination of ash content.

  • Bulgarian State Standard BSS EN ISO 4833-1:2013. Microbiology of the food chain – horizontal method for the enumeration of microorganisms – part 1: Colony count at 30°C by the pour plate technique.

  • Bulgarian State Standard BSS EN ISO 6579: 2003. Microbiology of food and animal feeding stuffs – horizontal method for the detection of Salmonella spp (ISO 6579:2002).

  • Bulgarian State Standard BSS EN ISO 6888-1:2005 + A1: 2005. Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) – part 1: technique using Baird-Parker agar medium – amendment 1: inclusion of precision data (ISO 6888-1:1999/Amd 1:2003).

  • Bulgarian State Standard BSS EN ISO 16649-2:2014. Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – part 2: Colony count technique at 44°C using 5-bromo-4-chloro-3-indolul beta-D-glucuronide.

  • Bulgarian State Standard BSS ISO 6611:2006. Milk and milk products – enumeration of colony-forming units of yeasts and/or moulds – colony-count technique at 25°C.

  • Damianova, S., Vasileva, N., Todorova, S., Stefanova, R. and Ga-neva, E. 2009. Obtaining of functional food products. I. Yogurt with oat meal. Proceedings of Rouse University, Bulgaria, Volume 48, pages 169–174. (In Bulgarian).

  • Domagala, J. and Wszolek, M. 2008. Effect of concentration method and starter culture type on the texture and susceptibility to syneresis of yoghurt and bio-yoghurts made of goat‘s milk. Zywnosc-Nauka, Technologia, Jakosc 15: 118–128.

  • Domagala, J., Wszolek, M. and Dudzinska, A. 2012. The influence of the fortification method and starter culture type on the texture and microstructure of probiotic yoghurts prepared from goat‘s milk. Milchwissenschaft 67: 172–176.

  • Fonden, R., Leporanta, K. and Svensson, U. 2006. Nordic/Scandinavian fermented milk products. In: “Fermented Milks”, 1st Edition (ed. A.Y. Tamime), Blackwell Science Ltd, Oxford, UK, pages 156–173.

  • Heino, A., Uusi-Rauva, J. and Outinen, M. 2010. Pre-treatment methods of Edam cheese milk. Effect on cheese yield and quality. Journal of Food Science and Technology 43: 640–646.

  • ISO 2446:2008. Milk – determination of fat content.

  • ISO 6731:2010 (IDF 21:2010). Milk, cream and evaporated milk – determination of total solids content (Reference method).

  • ISO 8968-1:2014 (IDF 20-1:2014). Milk and milk products – determination of nitrogen content – part 1: Kjeldahl principle and crude protein calculation.

  • Kondratenko, M.S. and Simov, J.I. 2003. “Bulgarian yoghurt”. Association of Dairy Processors in Bulgaria, Sofia, 262. (In Bulgarian).

  • Küçükçetin, A., Yagin, H., Hinrichs, J. and Kulozik, U. 2003. Adaptation of bovine milk towards mare’s milk composition by means of membrane technology for koumiss manufacture. International Dairy Journal 13: 945–951.

  • Kumar, P., Sharma, N., Ranjan, R., Kumar, S., Bhat, Z. and Jeong, D. 2013. Perspective of membrane technology in dairy industry: a review. Asian-Australasian Journal of Animal Sciences 26: 1347–1358.

  • Macedo, A., Pinho, M. and Duarte, E. 2012. Application of ultrafiltration for valorization of ovine cheese whey. Procedia Engineering 44: 1949–1950.

  • Mahdian, E. and Tehrani, M. 2007. Evaluation of the effect of milk total solids on the relationship between growth and activity of starter cultures and quality of concentrated yoghurt. American-Eurasian Journal of Agricultural and Environmental Sciences 2: 587–592.

  • Marafon, A.P., Sumi, A., Alcantara, M.R., Tamime, A.Y. and Oliveira, M.N. 2011. Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins. LWT – Food Science and Technology 44: 511–519.

  • Martìn-Diana, A.B., Janer, C., Peláez, I. and Requena, T. 2003. Development of a fermented goat’s milk containing probiotic bacteria. International Dairy Journal 13: 827–833.

  • Mehaia, M.A. 2005. Manufacture of fresh Labneh from goat’s milk using ultrafiltration process. Journal of Food Technology 3: 24–29.

  • Meletharayil, G.H., Patel, H.A. and Huppertz, T. 2015. Rheological properties and microstructure of high protein acid gels prepared from reconstituted milk protein concentrate powders of different protein contents. International Dairy Journal 47: 64–71.

  • Moreno-Montoro, M., Olalla, M., Giménez-Martínez, R., Bergillos-Meca, T., Ruiz-López, M., Cabrera-Vique, C., Artacho, R. and Miguel Navarro-Alarcón, M. 2015. Ultrafiltration of skim goat’s milk increases its nutritional value by concentrating nonfat solids such as proteins, Ca, P, Mg, and Zn. Journal of Dairy Science 98: 7628–7634.

  • Oliveira, M.N., Sodini, I., Remeuf, F., Tissier, J.P. and Corrieu, G. 2002. Manufacture of fermented lactic beverages containing pro-biotic cultures. Journal of Food Science 67: 2336–2341.

  • Ong, L., Dagastine, R., Kentish, S. and Gras, S. 2013. Microstructure and composition of full fat Cheddar cheese made with ultrafiltered milk retentate. Foods 2: 310–331.

  • Otaibi, M.A. and El Demerdash, H. 2008. Improvement of the quality and shelf life of concentrated yoghurt (labneh) by the addition of some essential oils. African Journal of Microbiology Research 2: 156–161.

  • Özer, B.H. 2006. Production of concentrated products. In: “Fermented Milks”, 1st Edition (ed. A.Y. Tamime), Blackwell Science Ltd, Oxford, UK, pages 128–155.

  • Reschke da Cunha, C., Viotto, W.H. and Viotto, L.A. 2006. Use of low concentration retentates in reduced fat “Minas Frescal” cheese manufacture: effect on composition, proteolysis, viscoelastic properties and sensory acceptance. International Dairy Journal 16: 215–224.

  • Shamsia, S.M. and El-Ghannam, M.S. 2012. Manufacture of Labneh from cow’s milk using ultrafiltration retentate with or without addition of permeate concentrate. Journal of Animal Production Advances 2: 166–173.

  • Sodini, I., Montella, J. and Tong, P.S. 2005. Physical properties of yogurt fortified with various commercial whey protein concentrates. Journal of the Science of Food and Agriculture 85: 853–859.

  • Tamime, A.Y. 2013. In: “Membrane Processing: Dairy and Beverage Applications”, 1st Edition (ed. A.Y. Tamime), Development of membranes processes, Wiley-Blackwell, USA, pages 4–158.

  • Tamime, A.Y., Saarela, M., Sondergard, A.K., Mistry, V.V. and Shah, N.P. 2005. Production and maintenance of viability of probiotic microorganisms in dairy products. In: “Probiotic Dairy Products”, 1st Edition (ed. A.T. Tamime), Blackwell Science Ltd, Oxford, UK, pages 39–72.

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  • View in gallery

    Scheme of laboratory equipment with a replaceable plate and frame membrane module 1: valve; 2, 3, 4: manometers; 5: replaceable plate and frame membrane module; 6: pump; 7: tank for initial solution; 8: cylinder for permeate.

  • View in gallery

    Microbiological status of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter ZD. VRR, volume reduction ratio.

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    Microbiological status of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter MZ2. VRR, volume reduction ratio.

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    Microbiological status of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starter 1CM. VRR, volume reduction ratio.

  • View in gallery

    Kinetics of titratable acidity of probiotic Bulgarian yoghurts (control and ultrafiltration retentates at VRR = 2 and VRR = 3) with starters ZD, MZ2 and 1CM. VRR, volume reduction ratio.