Development of new functional fermented product: mulberry-whey beverage

This study was conducted to produce and evaluate probiotic whey beverage during cold storage for 21 days. Five mixes of probiotic drinks were formulated for sweet whey (SW) and black mulberry (BM) juice. Physiochemical and microbiological properties as well as organoleptic evaluation were determined during cold storage at 4±1°C. The obtained results indicated that acidity of beverage treatments was gradually increased during cold storage. Meanwhile, the pH values and total phenolic contents were decreased gradually during cold storage. On the other hand, the antioxidant activity was increased by increasing the percentage of black mulberry juice; however, it was decreased during cold storage. Results clearly indicated that the viability of L. rhamnosus GG & Bifidobacterium animalis ssp. lactis Bb-12 remained high up to 14 days then started to decline. Sensory properties of all samples were accepted while SWBM3 (25% whey with 75% BM juice) gained the highest organoleptic scores. Our results showed a high potential to produce and develop a new functional fermented whey product.


Introduction
In recent decades, the market for functional fermented milk components is continually increasing especially whey. As sub-product of the cheese industry, whey is widely used as food ingredients due to their nutritional composition (93% water, 5% lactose, 0.8% proteins, 9% minerals, and 0.36% fat) and the large volumes produced. Wheybased lactic beverages are a perfect way to reuse the liquid whey due to its high nutritional value, low cost and sensory value. [1][2][3] However, whey-based lactic beverages represent a part of non-traditional dairy products that need chemical, physical, and sensory properties for product development and quality control. 4 Several lactic acid bacteria (LAB) and yeast may be employed in lactic beverages production. Hence, lactic beverages may be taken into account as an important driver for the delivery of probiotic micro-organisms in the human gastro intestine. 5,6 Currently, many products are being developed that utilizing mix of whey, milk, fruit juice, probiotic bacteria and/ or yeast. 4,7-9 LAB has been extensively used in a wide range of the production of dairy fermented products. It could hydrolyze milk proteins; moreover, some of them can decompose β-lactoglobulin (BLG) "allergens" during life cycle in whey and milk. 10,11 Pescuma et al. 12,13 reported that the strains of Lactobacilus acidophilus, L. paracasei, and Bifidobacterium have the ability to analyse the BLG allergenic epitopes in vitro.
Mulberry (Morus alba L. or Folium mori) is the fruit of the mulberry tree belonging to the genus Morus of the Moreaceae family. This fruit is cultivated in Africa, Asia, Europe, North and South America. It is famous in China for medical treatment due to its high content of nutritive compounds such as vitamins, amino acids, minerals, phenolic acids, anthocyanins "cyanidin-3-glucoside and cyanidin-3rutinoside" and flavonols. [14][15][16][17] In addition to their numerous functional properties including anticancer, antidiabetic, antioxidant, antiinflammatory, and anti-aging, also decreases the risk of coronary heart disease obesity and diabetes. 15 The antioxidant properties of phenolic compounds have the ability to act as metal chelators, proton donors and reducing agents. 18 Mulberries contain alkaloids that activate macrophages which are white blood cells that stimulate the immune system, putting it on high active alert against health threats. 19 As predominant polyphenols, anthocyanins up to ∼3000 mg/kg FW are found in mulberry fruits, which is one example of important naturally derived antioxidants. These fruits have much higher anthocyanins content than blackcurrant, redcurrant, blueberry, and blackberry. 20 Although the abovementioned health benefits and antioxidant properties of mulberry fruits, fresh mulberry are soft and perishable fruit that possess a very short shelf-life, moreover, it has a low acidity. Hence, the aim of this study was to investigate the possibility of introducing functional whey-mulberry beverages in order to enhance the shelf-life, nutritional and organoleptic qualities. Influence of addition of mulberry fruits with different ratio on physiochemical, microbiological and sensorial characteristics of sweet whey (SW) was discussed during storage. Results of this investigation could be an indicator for the development of health promoting food ingredients.

Materials
Fresh whey from manufacturing of Ras-cheese was provided by the agricultural secondary school (Shibin El-Kom, Menoufia, Egypt). The chemical composition of whey consists mainly from total solids (6.3± 0.026 g/100 g), protein (0.8± 0.015 g/100 g), fat (0.5± 0.115 g/100 g) and lactose (4.5± 0.104 g/100 g). The whey was then subjected to heat treatment at 68°C for 15 min then stored in plastic bottles at 4°C after cooling in an ice bath and frozen for further analysis. Black mulberry (BM, Morus alba L.), skim milk powder and sugar were purchased from a local market located in Menoufia, Egypt.

Preparation of cultures
A pre-starter culture (PSC) was dissolved and diluted in 50 mL skimmed milk before sterilization at 121°C for 10 min, and then cooled to 5°C. The pre-cultures were warmed at 42°C for 20 min before inoculation. Starter cultures were prepared by transferring PSC in SW before experimental use; PSC (2% v/v) was used as inocula. Fermentations were performed in sealed bottles containing 300 mL of SW and incubated at 37°C until the pH value reached ⁓ 4.5, and the pH was then adjusted to 5.5 with 5N NaOH. All treatments of SW were subjected to heat treatment at 60°C for 10 min to stop the fermentation process by starter culture.
B. lactis Bb-12 and L. rhamnosus GG probiotic cultures were grown initially for 48 h followed by sub-culturing and incubating for a further 17 h. The culture was harvested after centrifugation at 3200 ×g, for 10 min at 4°C. The pellets were washed twice in one-quarter-strength Ringer's solution and concentrated 10-fold in the same diluent. A 1% inoculum of probiotic culture was distributed into the BM juices to get a final concentration of approximately 10 8 CFU mL -1 .

Preparation of mulberry-whey beverages
Five different whey based beverages (WBB) were prepared without BM (SW) and with 25% BM (SWBM1), 50% BM (SWBM2), 75% BM (SWBM3) and 100% BM (SWBM4), respectively (Figure 1), considering the volume of inoculum and sugar. Sugar was added at a concentration of 15% (w/v) of the probiotic whey based beverages. The resulting mixtures were formulated under aseptic condition then stored at 4°C for 21 days.

Microbiological analysis
At appropriate intervals over several days/weeks depending on the specific strain, viable cell counts were recorded before cold storage. Serial dilutions were subsequently performed and bacteria were counted by applying the standard plate count method. The counts of Lactobacillus bulgaricus were estimated by culture in MRS agar under anaerobic incubation at 37ºC for 2 days. M17 agar was used for the enumeration of S. thermophilus. The petri dishes were incubated aerobically for 2 days at 37ºC. 21 Enumerations of L. rhamnosus GG and bifidobacteria were carried out on MRS agar adjusted to pH 5.4 with glacial acetic acid under aerobic incubation at 37ºC for 48 h. The selectivity of the growth conditions was confirmed by microscope appearance of the cells from single colonies.

Determination of pH values and titratable acidity
The pH values of raw materials (BM and whey) and samples were determined using a digital pH meter (HI8519, Hanna Instruments, Italy) with a temperature compensator. The titratable acidity was determined according to the Association of Official Analytical Chemists. 22

Determination of total solids, soluble solids, protein, fat, lactose and ash content
The total solids content was measured by the oven drying method at 105ºC up to a constant weight. 22 The concentrate was characterized for lactose, ash and total soluble solids, according to the A.O.A.C. 23 Protein content was determined by the macro-Kjeldahl 24 and fat by the Gerber method. 25

Determination of total phenolics
The total phenolic content (TPC) of samples was determined by using Folin-Ciocalteu method according to López-García et al. 26 , with some modifications. Briefly, 0.1 mL of sample was mixed with 0.75 mL of diluted Folin-Ciocalteu reagent (10-fold dilution with distilled water). After 5 min of incubation at room temperature, 0.75 mL of 2% sodium carbonate solution was added to the mixture. Then, the mixture was incubated for 15 min at room temperature and absorbance was measured at 760 nm. The results were indicated as milligram of gallic acid equivalent per 100 mL of sample. Calibration curve was carried out with gallic acid aqueous solutions (8-80 µg/ mL).

Determination of antioxidant activity
The antioxidant activity of whey beverages was determined by DPPH assay. Briefly, 2 mL of the whey beverages in clean and labeled test tubes dissolved in methanol was mixed with 2 mL of 0.2 mM DPPH. The mixture was then homogenized and left at room temperature in the dark for 30 min. The absorbance by using a UV-VIS spectrophotometer (6705B0, Jenway, Norfolk, USA) was measured at 517 nm and methanol used as a blank. The DPPH radical scavenging activity was calculated as follows: Here, A is the absorbance of the DPPH solution without sample and B is the absorbance of solution containing sample.

Determination of anthocyanins
By the pH differential method, the total anthocyanins content of all beverages were measured according to Klopotek et al. 27 using a UV-VIS spectrophotometer (6705B0, Jenway, Norfolk, USA). The procedure involved extraction of the anthocyanin with ethanol and HCl instead of methanol and formic acid, respectively. The absorbance of the beverages was measured at 510 and 710 nm using buffer solutions (pH 1.0 and 4.5). The total anthocyanins content were calculated as follows: The results were expressed as mg malvidin-3-glucoside/100 mL (%).

Sensory evaluation
Sensory evaluation was conducted according to Dubost et al. 28 with several modifications. A group of ten trained panelists aged between 25 and 60 years old from the Food Technology Research Institute (CFTRI) were included to perform the sensory evaluations.
Panelists were asked to evaluate the samples for acceptability of flavor, sweetens, color and appearance via scoring the samples from 1 to10. All panelists were selected based on willingness to consume milk beverages with no negative allergic reactions. Samples were presented to the panelists in a plastic coded cup (50 mL) and the presentation order was randomized for each panelist. Water cups were provided between samples to cleanse the palate.

Statistical analysis
Statistical analysis of the data was carried out by ANOVA using SAS (Statistical Analysis System) software version 9.1.3. The significant differences among means were assessed by Duncan's multiple range test at p < 0.05.

Chemical composition of raw materials
The chemical composition of the SW and BM (%) are shown in Table 1. The results showed that the moisture content of BM was 87.40% and SW was 93.70%. Ash content was 3.48% and 0.5% in BM and SW, while fat content was 1.25% and 0.5% in BM and SW, respectively. Further, the protein content of BM was 1.23%, while in SW was 0.8% (Table 1). On the other hand, total soluble solids, total solids and acidity were 8.23 %, 12.60% and 0.65% in BM, while 5.70%, 6.30% and 0.32% in SW, respectively. Therefore, it could be noticed that BM recommended for processing due to its relatively high total soluble solids (TSS) and total solids (TS) contents, and SW is employed as a functional food ingredient because of its nutritional value.

pH values and acidity
The pH value of the whey beverage decreased significantly (P < 0.05) during cold storage after 21 days (Figure 2), due to the persistent metabolic activity of LAB to produce lactic acid and galactose by consumption of lactose during cold storage. Also, total acidity was increased gradually by increasing mulberry percentage at zero time (Fresh) of storage. The fermented SWBM1, SWBM2, SWBM3, and SWBM4 produced a higher amount of lactic acid compared with that prepared without BM (SW). As shown in Figure 2, the total acidity basically ranked in order SWBM4 > SWBM3 > SWBM2 > SWBM1 > SW after 21 days. It's probably due to the increase of anthocyanin content. The pH values and titratable acidity of black mulberry whey beverages during cold storage at 4±1°C.

Viability of probiotic microorganisms in wheymulberry beverages
There is no doubt that probiotic strains selected for commercial use keep their viability and functional activity throughout the shelf-life of whey beverage products. On this base, the commercial strain L. rhamnosus GG was identified as a versatile culture because of their capacity to remain viable in the low pH juice matrix at commercially acceptable levels for at least 12 weeks. 29 Data in Table 2 show a decrease in cell count was observed for all strains after 21 days. The viable cell count of L. rhamnosus GG was increased through the second week of storage for all treatments, then slightly decreased at end of storage. B. lactis Bb-12 is recognized as being more robust when confronted with hostile conditions. 30 B. lactis Bb-12 showed no significant decrease in viability over three weeks of storage in juice and the numbers remained above 10 6 CFU mL -1 at different pH degrees. Notably, survival of B. lactis Bb-12 & L. rhamnosus GG may be due to phenolic compounds that act as shell against oxygen, besides nutrients in whey. These results are in agreement with Matsumoto et al. 31 and Sheehan et al. 29 Values with different letters in the same column are significant different at P < 0.05

Anthocyanin content
The effect of adding different ratios of BM juice on anthocyanin content of whey beverages during cold storage is illustrated in Table  3. The total anthocyanin of mulberry whey beverages was increased by increasing the addition ratio of mulberry juices at zero time (Fresh) of storage. On the other hand, data showed a slight decrease in anthocyanin contents during cold storage. This slight decrease could be due to the influence of fermentation condition that affects the decay of anthocyanin. This result is in agreement with Viljanen et al. 32 As shown in Table 3, SWBM4 had the highest degradation level (about 17%) during cold storage, which contains 100% BM, but with reducing BM level in all treatments, degradation level reduced by about 5%. These results explain that ascorbic acid correlated with anthocyanin decay level. Our data is in agreement with Chung et al. 33 who reported that vitamin C (L-ascorbic acid) is known to accelerate anthocyanin degradation. Castañeda-Ovando et al. 34 reported that whey protein could improve anthocyanin stability by interacting with either the anthocyanin itself or with the ascorbic acid, this phenomenon is known as copigmentation.

Total phenolic content
The changes in TPC (equivalent mg gallic acid /100 mL) of BM whey beverages during cold storage at 4±1ºC are shown in Figure 3. TPC of whey beverages prepared with different ratios of BM juices was increased by increasing the concentration of BM juices.

Antioxidant activity
The effect of adding different ratios of BM juice on DPPH-radical scavenging activity (RSA) of whey beverages during cold storage at 4±1ºC were illustrated in Figure 4. DPPH radical scavenging activity (RSA) of all treatments (SWBM1, SWBM2, SWBM3, and SWBM4) has higher antioxidant activity than control (SW), and this was directly proportional to the percentage of added juice. DPPH radical scavenging activities of fermented mulberry whey beverages ranged from 20.60 to 94.40% at time zero. Where, DPPH radical scavenging activities were ranged from 7.80 to 50.40% after 21 days. These results are in agreement with Kim et al. 19 and Wang et al. 36 who they reported that fermented Noni juice exhibited lower antioxidant activity, reducing activity, and DPPH scavenging effect compared with unfermented Noni juice. A positive correlation was found between TPC and DPPH radical scavenging activity (r=0.956, P<0.05).

Sensory evaluation
Sensory evaluation of mulberry whey beverages prepared with 25.0, 50.0, 75.0 and 100.0% BM juices during cold storage are shown in Figure 5. The mulberry whey beverages prepared with 75.0% mulberry juice (SWBM3) gained the highest score for color, appearance, and flavor at zero time (Fresh) of storage and after 21 days of storage at 4 ±1ºC. Therefore, these samples came in the first order on comparison with other prepared beverages (SW, SWBM1, SWBM2, and SWBM4) up to 21 days of storage. SWBM3 was the most acceptable sample followed with SWBM2 and SWBM4, while SW and SWBM1 were the lowest acceptable samples since they had been given the lowest scores. Where, all treatments (SWBM2, SWBM3, and SWBM4) kept strong color body and no sedimentation in all treatments.

Figure 3
The TPC of black mulberry whey beverages during cold storage at 4±1°C. Figure 4 The changes in DPPH radical scavenging activity (%) of black mulberry whey beverages during cold storage at 4±1°C.

Figure 5
The sensory evaluation of black mulberry whey beverages during cold storage at 4±1°C.

Conclusion
The present study described development of new functional fermented product (mulberry-whey beverage). The results showed that the acidity, antioxidant activity, total phenolic, and anthocyanins contents were increased by increasing the percentage of black mulberry juice. Where, pH values were decreased. Our results indicated that the viability of L. rhamnosus GG & Bifidobacterium animalis ssp. lactis Bb-12 remained high up to 14 days then started to decline. SWBM3 samples (25% whey with 75% BM juice) gained the highest organoleptic scores. The beneficial effect for the health of black mulberry (anthocyanins), their incorporation in food and beverages industries will represent an extra important value in the near future.