Bacterial Strains as Vitamins Supplements to Prepare Functional Dairy Beverages

MATERIALS AND METHODS

Main materials: Papaya fruits (Carica papaya L.) were obtained from Horticultural Research Institute, Agriculture Research Center, Giza, Egypt. However fresh sweet whey was obtained from Faculty of Agricultural, Cairo University, Egypt.

The bacterial cultures are Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium bifidum separated or mixed by (1:1:1) in the form of freeze-dried culture. They obtained from Dairy Department, Microbiology Laboratory, National Research Centre, Egypt. They prepared as the mother culture by adding 1% of lyophilized cell culture into sterilized reconstituted skim milk powder (12%) and incubated at 42°C for 4-6 h.

Experiments
Preparation of papaya pulp: Papaya fruits were carefully washed, cut into pieces and blended, then filtered through a fine cloth. The mixed pulp was heated at 90°C for 3 min to inactivate pectin enzymes, after that they cooled rapidly to 15°C and filled into glass bottles. The fruit pulp were stored at -18°C until used¹².

Preparation of probiotic papaya-whey beverage: Homogenized Papaya pulp and sweet whey were mixed in proportion 40:60 (w/v) as advised by Mohamed et al.¹². Carboxyl methylcellulose (0.2%), citric acid (0.1%) and potassium sorbets (0.1%) were supplemented. The total soluble solids content was adjusted to 18.0% in all blends using sucrose. The blends were poured in 100 mL five glass bottles. They closed and pasteurized at 65°C for 30 min. The first bottle was serve as control (c), whereas the other four bottles (T1, T2, T3 and T4) were individually fortified (under sterilized conditions) with 1% Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium bifidum separated or mixed (1:1:1), respectively. The inoculated samples were incubated at 42°C until pH 4.7, then cooled to 5±2°C. The resulting beverages were examined for bacterial counts fresh and after 1 month of storage at 5±2°C. The gross chemical composition of fresh beverages was as follow: TS were 18.0%, lactose content was 2.81 mg/100 g, while titratable acidity was 0.30%.

Methods
Sensory evaluation: Twenty expert judges were selected from staff member of Dairy Department., National Research Center, Egypt, to evaluate flavor (60 points), appearance (30 points) and color (10 points) of the dairy beverages samples according to Deka et al.¹³.

Determination of bacterial counts: Lactobacilli counts were determined using MRS agar (Oxoid) according to De Man et al.¹⁴ and Bifidobacterium bifidum was determined according to Blanchette et al.¹⁵ using modified MRS agar (Oxoid) supplemented with 0.05% L. cysteine-HCL (Merck, Germany). Plates were incubated at 37°C for 48 h under anaerobic conditions (BBL Gas Pak, Becton Dickinson, Cockeysville MA, USA).

Determination of vitamins contents: Vitamin B-group content is estimated as mentioned by Sharaf et al.⁷ using HPLC technique, while vitamin E was determined according to the methods described by Pyka and Sliwiok¹⁶. Vitamin A was determined according to the methods described by Noll¹⁷.

RESULTS AND DISCUSSION

Organoleptic properties of papaya whey beverages: Organoleptic evaluation of the fresh resultant beverages showed no clear differences in all examined items (flavor, appearance and color) in all samples and all examined parameters were accebtable.

Bacterial count of the probiotic in beverages: Figure 1 showed the number of bacterial counts in fresh and stored papaya-whey-beverages. It could be concluded that the log count of probiotic strains were slightly decreased from the initial time (after incubation) to ted end of storage period (after one month) except L. rhamnosus which was slightly increase. This result may be due to the sensitivity of these strains to acid developed during the month of storage. These results were agree with the results of De Souza Oliveira et al.¹⁸ and El-Batawy et al.¹⁹.

Vitamins content: One of the multiples benefits that probiotics have is the capacity to synthesize vitamins. We have kenned for sometime that commensal bacteria engender vitamins, concretely B vitamins and play a major role in meeting our desiderata for these essential nutrients.

Figure 2-6 revealed the capability of the bacterial strains in syntheses of hydrophilic and hydrophobic vitamins. The present results revealed that L. rhamnosus had the highly ability in production of vitamin E, D, B2 and B12 where their values were: 18.042, 463.5, 1880 and 827.7 μg/100 mL sample, respectively compared to 2.315, 33.387, 5.378 and 338.1 μg/100 mL for control sample. On the other hand, L. acidophilus produced the highly amounts of vitamin A, K, nicotinic and thiamin where their values were 1551.4, 97357, 9829 and 23694 μg/100 mL sample respectively in comparison to 0.0281, 3331, 50.85 and 61.60 μg/100 mL for control samples. However, Bifidobacterium bifidum had the highest ability for production of Folic acid. It produced 315.5 μg/100 mL sample rather than all other strains. Using of 1% of mixture of the three-mentioned strained lead to decrease the values of all estimated vitamins except vitamin C. This may be due to the competition between others. Jones et al.²⁰ reported that bacteria in the gut engender vitamin K and recent evidence produce vitamin D is as well. While, Eck and Friel¹ mentioned that in vitro and studies in humans have documented the capacity of some probiotic strains to synthesize vitamin K, folic acid, vitamin B2 and B12.

Fig. 1:

Log count of probiotic strains during storage period in papaya-whey beverages

Fig. 2:

Different vitamin contents produced by different species of probiotic bacteria in papaya whey beverages

Fig. 3:

Capability of L.acidophilus strain for production of different vitamins in papaya-whey beverage (T1)

Fig. 4:

Capability of L. rhamnosus strain for production of different vitamins in papaya-whey beverage (T2)

Fig. 5:

Capability of Bifidobacterium bifidum strains for production of different vitamins in papaya-whey beverage (T3)

Fig. 6:

Capability of mixed strains (1:1:1) for production of different vitamins in papaya-whey beverages (T4)

In 2006, Thijssen et al.²¹ explained that there are two different forms of vitamin K and K1 (Phylloquinone) present in all photosynthetic plants and menaquinons and vitamin K2 which is primarily of bacterial origin.

Le Blanc³ reported that B-groupvitamins can be successfully produced by lactic acid bacteria, while Rossi et al.⁴ and Eck and Friel¹ reported that several folate-producing strains have been selected within the genus Bifidobacterium.

CONCLUSION

It be concluded that, some lactic acid bacteria can be successufully used to produce vitamins in papaya-whey bevarege to produced fanctial product and improve health.