APTEFF, 46, 1-269 (2015) UDC: 637.146.1/.3:638.178.2 DOI: 10.2298/APT1546045G BIBLID: 1450-7188 (2015) 46, 45-54 Original scientific paper PHYSICOCHEMICAL PROPERTIES OF HONEYBEE POLLEN ENRICHED ACIDOPHILUS MILK AND PROBIOTIC YOGHURT Jovana R. Glušac1*, Milka J. Stijepić1, Spasenija D. Milanović2 and Dragica M. Đurđević-Milošević3 1 School of Applied Medical Sciences, Nikole Pašića 4a, 79101 Prijedor, Bosnia and Herzegovina 2 University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia 3MP Lab, MP Bio d.o.o., Prokupačka 41, 11000 Belgrade, Serbia The aim of this work was to examine the possibility of preparation of acidophilus milk and probiotic yoghurt by processing of milk (1%, 2.8% and 3.2% w/w fat) enriched with honeybee pollen (0.6% w/w). The quality of produced fermented milks was followed by comparing pH value during fermentation and storage time, as well as of lactic acid con- tent and sensory properties during 14 days of storage. Fermentation time was influenced more the type of the starter culture than by honeybee pollen addition or fat level. The addition of honeybee pollen increased the production of lactic acid, regardless of the fat level. The effects of different starters (L. acidophilus LA-5, S. thermophilus and L. Del- brueckii ssp. bulgaricus) on the production of lactic acid were also different, but not as obvious as that due to pollen addition. The obtained results revealed that honeybee addi- tion had positive effects on the physicochemical and sensory properties of produced aci- dophilus milk and probiotic yoghurt. KEY WORDS: acidophilus milk, probiotic yoghurt, honeybee pollen, physicochemical characteristics INTRODUCTION Among many fermented dairy products, the most notable fermented milks are yoghurt, kefir, kumiss, acidophilus milk, buttermilk, and Bulgarian buttermilk. Recently, acidophilus milk is becoming one of the most desirable natural products for human nutrition due to its beneficial health effects (1). Furthermore, Lactobacillus acidophilus is the most commonly suggested organism for dietary use. All these positive findings have emerged for the consumption of acidophilus milk. Traditional acidophilus milk is made from low-fat milk, sterilized at 120oC for 15 minutes to stimulate growth of L. acidophilus. The high-heat treatment of milk is used to denature and release peptides from milk proteins, which helps the growth of L. acidophilus, due to its lack of a good proteolytic system for hydrolyzing milk proteins (2). Original acidophilus milk is a highly acidic product with no balancing * Corresponding author: Jovana R. Glušac, School of Applied Medical Sciences, Nikole Pašića 4a, 79101 Prijedor, Bosnia and Herzegovina, e-mail: [email protected] 45 APTEFF, 46, 1-269 (2015) UDC: 637.146.1/.3:638.178.2 DOI: 10.2298/APT1546045G BIBLID: 1450-7188 (2015) 46, 45-54 Original scientific paper flavours. However, because of its acidic flavour, most consumers do not generally relish it (3). Recently, honeybee pollen, as well as other apicultural products, has gained increased attention for its high nutritive value. Honeybee pollen is a rich source of protein (25%); essential amino acids; oil (6%), containing more than 51% polyunsaturated fatty acids (PUFA), of which 39% of linolenic, 20% palmitic and 13% of linoleic acids; more than 12 vitamins; 28 minerals; 11 enzymes or co-enzymes; 11 carbohydrates (35-61%); flavonoids and carotenoids; phytosterols (4). Honeybee pollen was found to have therapeutic properti- es (5,6). It is considered a nutrient-rich perfect food, and is promoted as a commercially available supplement (7). In recent years, the physiological functionality of natural and functional foods has re- ceived much attention due to the increasing interest in human health. As far as we know, the use of honeybee pollen as a food supplement in dairy products is rare. Therefore, the objective of the present work was to investigate the influence of the honeybee addition and different starter culture (LA-5 and probiotic) on the physicochemical and sensory properties of fermented milk products manufactured from milk with different fat content (1%, 2.8% and 3.2% w/w fat) after the production and during 14 days of storage. EXPERIMENTAL Materials The following milk samples: 1) 3.2 % fat content (3.2% proteins, 4.6% lactose, poly- unsaturated fatty acids), obtained from „VITALIA, IMLEK“, d.d. Mlijekoprodukt (Ko- zarska Dubica, Bosnia and Herzegovina); 2) 2.8% fat content (3.4% proteins, 4.6% lacto- se pH 6.82 ±0.02), obtained from MEGGLE Mljekara d.o.o. (Bihać, Bosnia and Herzego- vina; 3) 1% fat content obtained from the "d.d. Varaždin" (Croatia) were used for the pro- duction of fermented milk. Physical, chemical and microbiological characteristics of milk samples were entirely in accordance with the pertinent standards. Probiotic monoculture Lactobacillus acidophilus LA-5 (Chr. Hansen, Denmark) was applied to achieve a con- centration of 0.05g/l (w/v) in manufacturing fermented milks. Probiotic yoghurt contai- ning: L.acidophilus LA-5 was made in combination with yoghurt starter culture YC-X16 (Streptococcus thermophilus and L. delbrueckii ssp. bulgaricus, Chr. Hansen, Denmark) in the ratio of 4:1. Commercial polyfloral honeybee pollen (Prijedor, Bosnia and Herze- govina) was powdered and used in the concentration of 0.6% w/w in manufacturing both types of fermented milk products. Fermented milks manufacturing Milks with different fat content were heated at 95ºC for 10 min. The milk and bacteria mixture was incubated at 38ºC for 4 hours and the fermented milk formed was stored at 4ºC and used as a starter culture next day. Two series of yoghurt were made: 1) acidophi- lus milk (LA-5) without and with addition of honeybee pollen, and 2) probiotic yoghurts made with: LA-5 + yoghurt starter (YS) (LA-5 + YS-X16= 4:1), without and with pollen 46 APTEFF, 46, 1-269 (2015) UDC: 637.146.1/.3:638.178.2 DOI: 10.2298/APT1546045G BIBLID: 1450-7188 (2015) 46, 45-54 Original scientific paper addition. For each treatment, the pollen powder was dispersed in an aliquot of heat-trea- ted milk, and then mixed thoroughly with the remaining milk to achieve the desired con- centration. The milk was cooled to the optimal temperature (38oC), inoculated with the yoghurt starter-mother culture (50 ml) and incubated at the same temperature until pH 4.7 to 4.6 was reached. Fermentations were stopped by rapid cooling to 20oC and the fermen- ted milks were placed in a cold storage at 5oC ±1. Each trial was repeated three times. Methods After manufacturing, fermented milks were analyzed by measuring the pH value, lac- tic acid content and sensory properties. pH was measured using pH 510/mV Meter (Eu- tech Instruments, England) during fermentation and during 14 days of storage. Lactic acid content was calculated on the basic titratable acidity (8) during 14 days of storage. Sensory evaluation of yoghurt was profiled after the 1st and 14th day in the cold store. The sensory properties of yoghurt were evaluated by 5 trained panellists using the Internatio- nal Dairy Federation (IDF) method (9). The sensory attributes consisted of flavour, odo- ur, general appearance, colour and consistency, and the coefficients of significance (Fv) were: 2.4 for taste; 0.4 for odour; 0.2 for appearance and for colour and 0.8 for consisten- cy. Maximum score was 20, and the sensory scores were awarded for each attribute using a rating scale ranging between 1 and 5. The average value of 3 measurements was taken for further analysis. Values of different tests were expressed as the mean ± standard de- viation (x ± SD). All data were subjected to one-way ANOVA and means were compared by the Holm-Sidak test (SigmaPlot 11.0, Systat Software, Inc. USA). The level of signifi- cance was set at p < 0.05. RESULTS AND DISCUSSION Fermentation of all produced samples lasted between 3 and 10 hours (Fig. 1). The shorter fermentation time (about 4 hours) resulted in probiotic yoghurt samples compa- rable to acidophilus milks, regardless of the milk fat content. The addition of honeybee pollen caused a slight decrease in the fermentation time of probiotic yoghurt samples made from milk with the highest fat content (Fig. 2A), probably due to the presence of various carbohydrates, mainly glucose, fructose and sucrose (4). However, the fermentation time was influenced more by different types of starter cul- ture than by honeybee pollen addition or fat levels. As is evident from Fig. 1A, acido- philus milks had the longest fermentation time (9-10 hours), regardless of different fat level. According to the results of Iancu et al. (10), the strain of L. delbrueckii ssp. bulga- ricus is faster than the other lactobacilli in producing lactic acid, so this could explain the observed shorter fermentation time of probiotic yoghurt samples. 47 APTEFF, 46, 1-269 (2015) UDC: 637.146.1/.3:638.178.2 DOI: 10.2298/APT1546045G BIBLID: 1450-7188 (2015) 46, 45-54 Original scientific paper A) 1% fat 7.0 LA-5 LA-5+pollen LA-5+YS 6.5 LA-5+YS+pollen 6.0 pH 5.5 5.0 4.5 0246810 time (hours) B) 2.8% fat 7.0 LA-5 LA-5 + pollen LA-5+ S 6.5 LA-5+YS +pollen 6.0 pH 5.5 5.0 4.5 0246810 time (hours) C) 3.2% fat 7.0 LA-5 LA-5+pollen 6.5 LA-5+YS LA-5+YS+pollen 6.0 pH 5.5 5.0 4.5 0246810 time (hours) Figure 1. Fermentation time of acidophilus milk and probiotic yoghurt made of milk with 1% (A), 2.8% (B) and 3.2% fat (C) enriched with honeybee pollen (0.6%) 48 APTEFF, 46, 1-269 (2015) UDC: 637.146.1/.3:638.178.2 DOI: 10.2298/APT1546045G BIBLID: 1450-7188 (2015) 46, 45-54 Original scientific paper 5.0 A) 1% fat LA-5 LA-5+pollen 4.8 LA-5+YS LA-5+YS+pollen 4.6 pH 4.4 4.2 4.0 1 7 14 storage time (days) B) 2.8% fat 5.4 LA-5 LA-5+pollen 5.2 LA-5+YS LA-5+YS+pollen 5.0 pH4.8 4.6 4.4 4.2 4.0 1 7 14 storage time (days) 5.0 C) 3.2% fat LA-5 LA-5+pollen 4.8 LA-5+YS LA-5+YS+pollen 4.6 pH 4.4 4.2 4.0 1 7 14 storage time (days) Fiure 2.