Probiotic stability of yoghurts during refrigerated storage

Probiotic strains were isolated from the cheese, turkey and dimity and fermented products (rayb, zeer milk,kariesh cheese), screened for clotting time of skim milk. The seven effective isolates which clotting skim milk were identified as Bifidobacterium spp (RC1 b8, RC4 b2, FC1b1, SC1 a4, LZ1b8 and LZ1a7)and Lactobacillus spp (S4b1). The seven effective strains of probiotic bacteria were used in the production of probiotic yoghurt 100%, yoghurt of 50% probiotic bacteria with 50% yoghurt bacteria and compared with the normal yoghurt with 100% normal yoghurt bacteria. The changes in pH, stability of bacteria, and physical properties in probiotic yoghurt 100%, yoghurt of 50% probiotic bacteria with 50% yoghurt bacteria and the normal yoghurt with 100% normal yoghurt bacteria during product’s shelf life at 4C for four weaks were also studied.


INTRODUCTION
Many probiotic bacteria used today in yoghurt grow poorly in milk when compared with some common starter cultures, such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus.Also, probiotic strains do not survive well in the final yoghurt product (Schillinger, 1999), or may even change the traditional taste of yoghurt.Although there are no set standards concerning the population of the probiotic organism in yoghurt at the end of the products shelf life, it is usually considered that anywhere between 5 and 8 log cfu g_1 is an acceptable final population (Shah et al, 1995;Schillinger, 1999;Svensson, 1999).Probiotics are live microorganisms which transit the gastrointestinal tract and in doing so benefit the health of the consumer (Tannock et al., 2000).Therapeutic benefits have led to an increase in the incorporation of probiotic bacteria such as lactobacilli and bifidobacteria in dairy products, especially yoghurts (Lourens -Hattingh and Viljoen, 2001).There were significant strain differences in the viability of probiotic bacteria during storage of cultured dairy products (Ross et al., 2002).
Probiotics such as Lactobacillus and Bifidobacterium spp.are bacterial members of the normal human intestinal flora (Tamime et al., 2005).Several reports have shown that survival and viability of probiotic bacteria is often low in yoghurt and results in less than 108-109 cells daily recommended intake (Lourens-Hattingh and Viljoen, 2001).The suggested concentration for probiotic bacteria is in the range 106-107 cfu/g of the product (Robinson, 1987and Salminen et al.,1993).
Dairy products such as fermented milk and yoghurt are often used as carriers for probiotic cultures.
It was shown for the first time that probiotic and other lactic bacteria exhibit an antiviral activity in a cell culture model.Possible mechanisms of antiviral activity include: 1) hindering the adsorption and cell internalisation of the VSV due to the direct trapping of the virus by the bacteria, 2) "crosstalk" with the cells in establishing the antiviral protection and 3) production of metabolites with a direct antiviral effect, Botić et al.,(2007).
High levels of viable microorganisms are recommended in probiotic foods for efficacy (Knorr, 1998).To achieve the claimed health benefits, one of the most important requirements for manufacturing and marketing of probiotic yoghurt is to maintain a high number of probiotic organisms P6 log CFU/g at the point of consumption (Lourens-Hattingh and Viljoen, 2001).Donkor et al., (2006) reviewed the factors that may affect the survival of Lactobacillus and Bifidobacterium spp.In yogurt: strain of probiotic bacteria, inoculation level, incubation temperature, pH, growth promoters and inhibitors, presence of hydrogen peroxide and oxygen, concentration of metabolites, lactic and acetic acid, buffering capacity of the media, storage temperature, and availability of nutrients.The main factors are ultimate pH and accumulation of organic acids (Dave and Shah, 1997and Shah, 2000).
Co-culturing with proteolytic yoghurt bacteria i.e.Lactobacillus delbrueckii subsp.bulgaricus (LB) and Streptococcus thermophilus (ST) also enhances the growth and viability of probiotics and helps to reduce fermentation time (Dave and Shah, 1997).To support the growth and survival of probiotic bacteria in fermented dairy products.
The aim of the present work to use the isolated probiotic bacteria which had strength of clot skim milk in the production of probiotic yogurt, Ali et al.,(2010 and2012), to examine the changes in pH, stability of bacteria, and physical properties of seven probiotic strains in yoghurt alone or with 50% normal yoghurt bacteria compared with 100% normal yoghurt bacteria during product's shelf life at 4 ْ◌ C.

MATERALS AND METHODS Yoghurt production:
For set-type yoghurt production, cows' milk was heated at 60-75C, homogenised under pressure of 100-200 bar, pasteurised at 85-95 ْ◌C and cooled at 37 to42 ْ◌C.After inoculation with the appropriate inoculum type, milk was distributed to 100mL plastic retail containers, sealed, incubated at 37 to 42 ْ◌C.until pH reached 4.6, and then cooled and stored at 4 ْ◌C.At time intervals of 1, 7, 14,21and 28 days, yoghurt samples were subjected to microbiological, and physicochemical analysis.All yoghurt trials were repeated in twice.Types of inocula and conditions used are shown in tables(1 to 4).When curdled milk inoculum was used, the milk was inoculated at 2% (v/v) with each strain and incubated at 37 or 42 ْ◌C.
In each case the starter cultures were combined with one of the adjuncts.In addition, yoghurt was prepared with a 1% (v/v) inoculum and 37 to 42 ْ◌C incubation temperature, using the same starter cultures and 1% (v/v)  The inoculated milk samples were aseptically transferred into 100 ml plastic containers,tightly sealed, incubated at 40 ْ◌C overnight and transferred to fridge at 4 ْ◌C the following day.All trials were replicated three times.The sampling schedule for testing was immediately after the addition of starter culture into milk (day 0), after overnight incubation (day 1), fermentation and at weekly intervals (days 7, 14, 21 and 28).The analyses were performed on triplicate samples for pH, and in duplicate for bacterial counts, firmness of yoghurt gel (curd tension)and syneresis.

Analytical determinations of set yoghurt: Determination of pH:
The pH of yoghurt was measured using a bench-top pH-meter which was previously calibrated with pH 7.0 and 4.0 standard buffers.

Enumeration of probiotic and starter cultures:
Ten gram samples of each yoghurt batch were diluted with 90 ml of 0.1% sterile buffered peptone water (Oxoid Ltd.,Hampshire, UK) and placed in stomacher for 2 min.Tenfold serial dilutions of 10_2-10_9 were then prepared in 9 ml of 0.1% sterile peptone water and 1 ml of the three highest dilutions was pour-plated in duplicate.Colonies count of strains were enumerated on MRS agar under anaerobic condition at 30 ْ◌C for 72 h.The numbers of colony forming units (CFU) on plates containing 25-250 colonies were calculated per gram of sample.

Firmness of yoghurt gel (curd tension):
The firmness of the formed yoghurt gel was expressed as the weight (gm) required for a plastic tube to penetrate into the gel from surface to bottom.Syneresis: Amount of whey drained(ml/100gm of yoghurt within 30 minuts).

Statistical analysis:
All results were statistically analysed using Duncan's Multiple Range test (DMRT,1955) was applied for mean comparison when one-way analysis of variance (ANOVA) showed significant differences at the 95%confidence level.

RESULTS a-Changes in pH during yoghurt storage:
Probiotic bacteria are slow acid producers (Marshall and Tamime, 1997).The yoghurt starter cultures including L. delbrueckii subsp bulgaricus and S. thermophilus are active even at refrigerated temperature and still can produce small amounts of lactic acid by fermentation of lactose which results is noticeable pH decrease (Shah et al., 1995).Several reports have indicated that there are significant differences between probiotic strains with respect to survival in acid environment.The problem of sensitivity to acidity of the probiotic culture is compounded by the fact that acidity may increase during storage, a phenomenon known as 'over acidification'.
The pH in the control (Y.S.) and the experimental yoghurt (7 bacterial isolates, Fig. 1, alone and mixed with Y.S. at 1:1) during storage at 4C for 4 weeks shown in Table (1).In fresh yoghurt, the control showed the lowest pH (3.4), followed by the treatments of the mixed isolates + Y.S., which exhibited slightly higher pH values (3.5 -3.7).In case of the isolates alone, only 3 isolates exhibited pH values < 4.0 (3.6 -3.9), while the rest isolates exhibited higher pH values (4.1 -4.6).During storage, the pH in all treatment remained stable or slightly increased till the 4th week, except for the treatments of LZ1b8, RC1b8 and their mixture with Y.S. Generally, it was obvious that the pH values for the treatments of the individual isolates were always higher than for the treatments of the isolates mixed with Y.S.
The control yoghurt with the traditional yoghurt starter cultures showed the lowest pH.This finding may explain the result of high pH values for the yoghurts based on the individual isolates.Also, the yoghurt starter cultures including L. delbrueckii var.bulgaricus and S. thermophilus proved to be active even at refrigeration and can produce small amount of lactic acid by fermentation of lactose which result in noticeable pH decrease (Shah et al., 1995).This statement also explains the lower pH values obtained in the control yoghurt and the treatments of mixed isolates with Y.S., where both contained L. bulgaricus and S. thermophilus.The initial pH prior to storage ranged from 4.2 to 4.5 that dropped to 4.1-4.3after 28 days (Paseephol.and. Sherkat., 2009).(1) Y.S. = Traditional starter contained standard yoghurt culture of Lactobacillus bulgaricus + Streptococcus thermophilus.

B-Bacterial culture stability in yoghurt:
There are significant strain differences in the viability of probiotic bacteria during storage of cultured dairy products (Ross et al., 2002).The viability of 7 bacterial isolates (Ali et al., 2012) either alone or in the presence of a traditional yoghurt starter (Y.S.) culture during storage of yoghurt at 4 ْ◌C for 4 weeks is presented in Table (2).
It is clear that the initial counts at zero time (immediately after inoculation) in all treatments were considerably higher (ranged 2.5x107 -1.1x109 cfu /ml) than the control (Y.S. = 5.6x106 cfu/ml).The yoghurt kept in refrigerator overnight (approx.24 hrs) exhibited a remarkable increase in the viable counts of all isolates alone and the control (almost 10-folds) as compared to the slight or no increase in the viable counts of bacterial mixtures (isolate +Y.S.).The presence of L. delbrueckii bulgaricus in Y.S. could be responsible for the reduced viable counts due to decreasing the pH (Table 1), in the mixtures treatments as compared to the yoghurt from the isolates alone.Rybaka and Kailasapathy (1995) reported that the presence of L. bulgaricus was found to be the main detrimental factor responsible for Bifidobacterium sp.mortality, and when L. bulgaricus was excluded from yoghurt manufacture, the decrease in pH was significantly reduced during storage, as L. bulgaricus cause over acidification during manufacture and storage.Also, Dave and Shah (1997)  (2) Probiotic isolate alone as a starter (in skim milk).( 3) Probiotic isolate mixed with Y.S. at a ratio of 1:1.(4) Means a column followed by the same letter (s) are not significantly different at 1% level of probability (Duncan's multiple-range test, Duncan, 1955).
In comparison, the isolates alone showed almost a similar pattern, where the counts increased variably till the 3rd week, followed by a considerable decline in the 4th week in most of the isolates.The viable counts for all the isolates alone remained always higher than the control.Besides, the isolates FC1b1 and SC1a4 alone gave the highest counts as compared to the other isolates throughout the whole period of storage.The major differences between the probiotics survival were related to species differences and there was little variance between different commercial strains of the same Bifidobacterium or L. acidophilus .All Bifidobacterium sp.survived well, as did L. casei, L. paracasei and L. rhamnosus.Lactobacillus strains (L26, Lc1 and DR20) showed survival patterns similar to the Bifidobacterium spp.The L. casei (Lc1) maintained at the initial levels and then decreased slightly by 32 weeks to be present at 1.6×107 CFU/g ( Phillips et al,2006).
As for the mixtures (isolates + Y.S.), the viable counts obviously increased in all treatments till the 3rd week, then the counts showed a slight increase or remained stable in certain cases, i.e.S4b1+Y.S., LZ1b8+Y.S. and RC1b8+Y.S., while a considerable decline in those counts occurred in the rest treatments.Also, no inhibition was evident from the two starter cultures against the lactobacilli strains.This allows the co-existence of the two starter cultures with the probiotics isolates in yoghurt ( Maragkoudakis et al., 2006).
In agreement with the present results, Haddadin et al. (2004) produced yoghurts containing counts of > 1.0 x 108 cfu / ml of the individual probiotics and high counts of the traditional species, and storage trials at 4 ْ◌C.showed that the viability of the probiotic cultures was retained over 15 days.It has been suggested that the probiotic bacterial population in a fermented milk product should be above 6 log cfu g_1 at the end of its shelf life, in order to exert any beneficial effect on the host (Shah et al., 1995;Schillinger, 1999and Svensson, 1999).Also, Linn et al. (2008) recorded counts of > 1.0 x 109 cfu / ml for the yoghurts based on the individual probiotics, and high counts for the traditional species were also obtained.Storage trials at 4 ْ◌C showed that the viability of the probiotic cultures was retained over 3 weeks then decreased to 2.8 x 106 on the 28th day.These observations were consistent with the findings of Akalin et al. (2004)and Dave and Shah (1997).Who reported higher stability of ST than LB and bifidobacteria in probiotic yoghurts during storage time.
In addition, Tabatabaie and Mortazavi (2008) concluded that the survival of the two probiotic strains L. rhamnosus and Bifidobacterium bifidum was found to be extremely stable during 5 weeks storage period at 4 ْ◌C.The initial counts of ST in all inoculated milks ranged from5.3 to 5.5 log CFU/ml that increased by ca. 3 log cycles after overnight incubation Paseephol, and Sherkat. (2009).
The experimental and control yoghurt s showed ability in sustaining high numbers of ST up to 14 days of storage (P > 0.05) and only a marginal decline occurred in the following 14 days.After 28 days, all yoghurts contained >8.0 log CFU/g of ST,decreasing from the initial counts by only 2.7-4.2%.This reflected the high stability of ST in the products Paseephol.andSherkat.(2 0 0 9).

C-Firmness of yoghurt gel (curd tension):
Table (3) shows the changes in the firmness of yoghurt gel during refrigerated storage for 4 weeks.In fresh yoghurt, the curd tension (C.T.) was higher for all treatments of the isolates mixed with Y.S. (53.0 -63.2 gm) than the control 52.4 gm, and the highest value was recorded for LZ1a7+Y.S. treatment.Meanwhile, the treatment of all isolates alone exhibited much lower C.T. values ranged (21.8 -41.1 gm), and the highest values was recorded for FC1b1 treatment.

15
A noticeable decline then occurred in C.T. in treatments and the control till the 2 nd week of storage.In the 4 th week, treatments of the isolates LZ1a7, LZ1b8, and their mixture with Y.S., as well as the control exhibited some increase in C.T., while the rest of treatments showed some decline in C.T.
The physical character of the curd is of primary importance with reference to the consistency of cultured yoghurt.In general, the obtained results of fresh yoghurt indicate that the curd tension values coincided greatly with the previous results of lower pH, and vice versa (Table 1).This is in agreement with Mohammad (2009) who concluded that the higher the pH level, the lower the curd tension occurred.It is also necessary to explain the changes in yoghurt during storage, the yoghurt gel is not a tight matrix but formed as a loose structure with fractal characteristics, however, post acidification over the storage period and consequently casein arrangement around the bacterial cells could result in a more compact and continuous structure (Hassan et al., 1996).

D-Syneresis:
Table (4) presents the syneresis (ml/ 100gm within 30 minutes) for all yoghurt treatments and the control.The syneresis, in fresh yoghurt, was 26.3 ml for the control as compared to (22.8-35.0ml) for the treatments of the individual isolates, and (21.0-29.3ml) for the treatments of isolates +Y.S. Measurement of syneresis fluctuated throughout the whole period of storage till the 4 th week, at which time a noticeable decline in syneresis occurred in most treatment.during storage at 4 ْ◌C.
It is well apparent, in the present results, that the ranges of syneresis for both the treatments of the isolates alone or mixed with Y.S. were almost closely LZ1b8 Zeer milk (Bifidobacterium spp.) Fig. 1: Bacterial shape of good clotting probiotic isolates.

Table 1 :
Changes in pH of yoghurts based on individual probiotic isolates or mixed with traditional yoghurt starter (YS) during storage at 4 ْ◌C.

Table 2 :
Stability of probiotic isolates or in the presence of a traditional yoghurt starter (YS) culture during the storage of yoghurts at 4 ْ◌C for 4 weeks.(1) Y.S. = Traditional starter contained standard yoghurt culture of Lactobacillus bulgaricus + Streptococcus thermophilus.

Table 3 :
Curd tension changes of yoghurt based on probiotic isolates alone or mixed with traditional starter during storage at 4 0 C.

Table 4 :
Syneresis of yoghurt based on probiotic isolates alone or mixed with traditional starter (YS)