Effects of Food Matrix and Storage Conditions on the Survival of Probiotics during Storage
Food matrix, storage conditions, and storage time significantly affected the survivability of B. longum
, L. acidophilus
, and L. plantarum
< 0.05). The average populations of the three strains in the samples reduced with increasing storage time (Table 1
). The reduction was significantly (p
< 0.05) higher at 30 °C 50% RH compared to the storage condition of 20 °C 20% RH. Foods used as carriers had a significant effect (p
< 0.05) on the survival of the strains on week 0, week 2, and week 4.
was initially coated onto all substrates at about log 7 cfu g−1
, except rice collet, which was significantly lower at approximately log 6.2 cfu g−1
). L. acidophilus
and L. plantarum
RC30 were coated onto all substrates with loads ranging from log 4.5 to log 6.3, and there was no obvious trend.
After two weeks of storage at either 20 °C 20% RH or 30 °C 50% RH, B. longum ATCC15707T survival reduced significantly (p < 0.05) when coated onto all substrates except for oat or wheat bran at 20 °C and 20% RH. After four weeks of storage at either 20 °C 20% RH or 30 °C 50% RH, only storage at 20 °C 20% RH on wheat bran did not show a significant reduction of cfu from time zero. After as little as two weeks of storage at 30 °C 50% RH, B. longum ATCC15707T survival on raisin had reduced to less than log 1 cfu g−1.
After two weeks of storage at either 20 °C 20% RH or 30 °C 50% RH, L. acidophilus ATCC4356T showed a significant (p < 0.05) reduction in survival when coated on all substrates. When L. acidophilus ATCC4356T was coated onto rice collet or raisin, no viable bacteria were recovered after two weeks of storage at 30 °C 50% RH.
L. plantarum RC30 significantly (p < 0.05) reduced on all substrates after two weeks of storage at 20 °C 20% RH, however it appeared to be stabilized with no further reduction for all substrates except coconut and raisin, which showed further significant (p < 0.05) reduction. However, there were not significant reductions of L. plantarum RC30 at 30 °C 50% RH at either week two or week four (p < 0.05). Indeed, no bacteria could be recovered after coating on raisin after 2 weeks.
Studying the survival of probiotic bacteria at 20 °C 20% RH and 30 °C 50% RH can provide useful information to evaluate the storage conditions and quality of non-refrigerated probiotic foods. We found that storage temperature and relative humidity have a significant effect on the survival of probiotic bacteria. The population of bacteria recovered from samples was higher at 20 °C 20% RH than that at 30 °C 50% RH, which is accordance with the results from other studies [19
]. Viability loss of B. lactis
BB12 at 30 °C has been shown to be related to water activity, as no viable bacteria were detected after 8 days of storage at aw
0.54 compared to 0.1% of viability loss with aw
0.33 after 2 weeks of storage [22
]. In addition, the viability of L. rhamnosus
GG formulated with flaxseed after storage for 14 months at 22 °C was reported to show a reduction of viability by more than 4 log cfu with aw
0.43, but a slight reduction of only 0.29 log cfu with aw
]. It has been shown that low water activity in a food carrier maintains the enzyme activity of bacteria during storage, which may contribute to improved survival [24
]. While the work reported here did not measure water activity of the samples, it is reasonable to estimate that the samples produced would have equilbriated to water activities of approximatley 0.2 and 0.5 at 20 °C 20% RH or 30 °C 50% RH, respectively. Low relative humidity (and therefore low water activity) has been shown to improve survival of L. rhamnosus
GG at 11% RH. However, this was dependent upon temperature, with less survival at 37 °C than at 25 °C [25
]. It has been suggested that high storage temperature accelerates metabolic and cellular activities of probiotics, resulting in depletion of nutrients and contributing to loss of viability and the oxidation of cell contents [26
]. Essentially, high temperature and high water activity (humidity) lead to reduced viability.
Under the storage condition of 20 °C 20% RH, the best survival of: B. longum
was on wheat bran; for L. acidophilus
on oat or wheat bran; and L. plantarum
RC30 coated on oat. At storage of 30 °C 50% RH, the best survival of all three bacteria was on peanut (and oat for L. plantarum
RC30). This is supported in the literature; peanut butter has been shown to protect probiotics such as Streptococcus
, and Bifidobacterium
]. This may be due to the buffering capacity of the fat in the peanut butter [27
]. Interestingly, we only observed this under the most adverse storage conditions (30 °C 50% RH). In addition, dietary fibers may also aid the viability of probiotics; for example, oat bran increased the stability of Lactobacillus casei
LC-1 at 10 °C, 25 °C, and 40 °C compared with inulin, unripe banana flour, and apple [28
]. This may be used to explain why oat generally provided a good protection for all three bacteria at 20 °C and 30 °C in this research. Clearly, the probiotic viability when formulated with foods is linked to species and type of food matrix [22
]. However, another study found that survival of L. rhamnosus
E899 and L. rhamnosus
E522 on oat in low pH apple juice at 20 °C was better than at 4 °C [29
]. This is contrary to more frequently reported results indicating that higher storage temperature results in poorer survival.
Although the initial population in the fresh culture for each bacterium was the same at ~log 8 cfu mL−1, the initial loading of the bacteria on foods was different and covered a range of ~log 5 to log ~7 cfu g−1. Since the method of sample preparation involved direct mixing of fixed quantities of materials, it can be assumed that all bacteria were loaded onto the food carriers. Clearly, something soon after coating contributed to a reduction in cell viability. This is likely related to both the bacteria species and type of food substrate.
It is interesting to note that B. longum
was the most stable probiotic bacteria during storage in the present study, which is contrary to a general suggestion that Bifidobacteria
are more sensitive to oxygen than Lactobacilli
due to their anaerobic nature [4
]. However, a study by Klu et al. reported that Bifidobacterium
had the greater survival at 4 °C, 25 °C, and 37 °C during storage over 12 months compared to Lactobacillus
]. The stability of L. acidophilus
has been reported to be affected by nutritional status, thus affecting its cell morphology—short cells of L. acidophilus
being more stable than long filamentous rods [15
]. While subjective, our microscopic observations (data not shown) suggested more long filamentous rods and less single short rods of L. acidophilus
after anaerobic incubation for three days at 37 °C. It is possible that the L. acidophilus
samples prepared for this investigation may have contributed to their poorer stability compared to Bifidobacteria