The Impact of Storage Conditions on the Stability of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb12 in Human Milk

Abstract

Human milk is the optimal source of complete nutrition for neonates and it also guides the development of infant gut microbiota. Importantly, human milk can be supplemented with probiotics to complement the health benefits of breastfeeding. Storage of human milk for limited periods of time is often unavoidable, but little is known about the effect of different storage conditions (temperature) on the viability of the added probiotics. Therefore, in this study, we evaluated how different storage conditions affect the viability of two specific widely used probiotics, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis subsp. lactis (Bb12), in human milk by culturing and quantitative polymerase chain reaction. Our results indicate that LGG and Bb12 remained stable throughout the storage period. Thus, we conclude that human milk offers an appropriate matrix for probiotic supplementation.

Introduction

Human milk is considered the gold standard in infant nutrition and has been identified as a continuous source of beneficial microbes and other microbiota modulating components, such as human milk oligosaccharides.^1,2 Very preterm or significantly ill neonates may not be able to feed orally or their mothers may not be able to provide their milk because of illness or separation and long distances. Human milk banks in hospital settings offer these neonates stored mother's milk or donor human milk. The use of human milk or probiotics has been associated with reduced risk of complications of prematurity, including necrotizing enterocolitis (NEC) and infections of the respiratory tract.^3–5 Interestingly, probiotic-supplemented human milk appears to confer better protection against NEC than nonsupplemented human milk.⁶ Human milk is, therefore, an attractive matrix for the delivery of probiotics, but there are still concerns about the safety and ability of different probiotic strains to remain stable during storage. No studies have to our knowledge been conducted to determine the viability of two of the most widely used probiotic strains, Lactobacillus rhamnosus GG (LGG) (ATCC 53013) and Bifidobacterium animalis subsp. lactis Bb12 (DSM15954), in human milk. These two probiotic strains have been documented for several health benefits in infants. Thus, their supplementation and stability in human milk could promote infant health. The aim of this study was to evaluate the viability of the two probiotic strains by culturing and quantitative polymerase chain reaction (qPCR) when supplemented in human milk and stored at room temperature, +4°C, and −20°C.

Materials and Methods

A total of eight human milk samples originating from an ethically approved study by our group were selected.⁷ The milk samples were pooled together and then divided into three groups: (1) supplementation with ∼1 × 10⁸ colony forming units (CFU)/mL LGG, (2) ∼1 × 10⁸ CFU/mL Bb12, and (3) no supplementation, that is, control. Both probiotic strains were obtained from Chr. Hansen (Copenhagen, Denmark). The stability of both LGG and Bb12 was assessed in triplicate, in three independent experiments. For supplementation, LGG was grown overnight aerobically in de Man Rogosa (MRS) broth (Oxoid, Basingstoke, United Kingdom) at 37°C and Bb12 in Gifu Anaerobic Medium (GAM broth) (Nissui Pharmaceutical, Tokyo, Japan) at 37°C under anaerobic conditions (10% H₂, 10% CO₂, and 80% N₂; Concept 400 anaerobic chamber, Ruskinn Technology, Leeds, United Kingdom). The bacteria were harvested by centrifugation (17.000 g, 5 minutes), washed twice, resuspended in phosphate-buffered saline buffer, and plated to create a standard growth curve. Supplemented samples were kept at room temperature (23°C), refrigerated condition (+4°C), and freeze-stored (−20°C) to assess the influence of the human milk matrix on the viability of both probiotic strains with storage (Table 1). The specific storage periods that were examined in this study were chosen in accordance with the milk storage guidelines.⁸

Table 1.

Storage Conditions of Milk Supplemented with LGG or Bb12

Temperature	Time
Room temperature	0 hours
Room temperature	6 hours
+4°C	6 hours
	24 hours
	72 hours
−20°C	7 days

LGG, Lactobacillus rhamnosus GG.

Serial dilutions were prepared and spread onto the MRS and GAM plates in triplicate and were incubated aerobically and anaerobically for 48 and 72 hours at 37°C, respectively. The enumeration data were expressed as log₁₀ CFU/mL. LGG and Bb12-like colonies were confirmed by colony PCR with strain-specific primers (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/bfm). The amplified DNA was then purified with polyethylene glycol 8000/NaCl (PEG 8000; AppliChem, Darmstadt, Germany) and sequenced with the ABI Prism-3130XL genetic analyzer (Applied Biosystems, Foster City, CA) by Turku Center for Biotechnology (Turku, Finland). The resulting sequences were identified by using BLAST searches (NCBI) against the GenBank database.⁹

For the qPCR analysis, total DNA was extracted using the InviMag Stool DNA kit (Stratec Molecular, Berlin, Germany) following a protocol as previously described by Kumar et al.⁷ The DNA standards for qPCR for LGG and Bb12 were prepared as previously described.¹⁰ Amplification of the DNA was performed with an ABI 7300 Real-Time PCR system (Applied Biosystems). The amplification program consisted of an initial cycle of 95°C for 3 minutes and 40 cycles of amplification (95°C for 5 seconds, 60°C for 30 seconds for LGG and 68°C for 30 seconds for Bb12), followed by a dissociation stage (Supplementary Table S1). qPCR amplifications were carried out in duplicate.

Results

Within the study periods, there were no statistically significant decreases in the number of viable cells of either LGG or Bb12 in any of the conditions studied (p > 0.05) (Fig. 1). Interestingly, the most notable decrease in the viable counts for both LGG and Bb12 was observed in supplemented milk that was stored at 4°C for 72 hours, with the detected decrease in viable cells 0.6 (standard deviation [SD] 0.3) and 0.3 (SD 0.2) log CFU/mL, respectively (Supplementary Table S2). However, this decline was not statistically significant. In addition to the probiotic bacteria used in the study, only two bacterial species identified as Staphylococcus epidermidis (211 CFU/mL) and Staphylococcus warneri (61 CFU/mL) were isolated from the nonsupplemented milk (control) using conventional culturing.

FIG. 1.

Average bacteria count of probiotic-supplemented human milk samples stored in different conditions. (A) Samples of human milk supplemented with LGG evaluated by conventional culturing. (B) Samples of human milk supplemented with Bifidobacterium animalis subsp. lactis Bb12 evaluated by conventional culturing. (C) Samples of human milk supplemented with LGG evaluated by qPCR. (D) Samples of human milk supplemented with Bifidobacterium animalis subsp. lactis Bb12 evaluated by qPCR. LGG, Lactobacillus rhamnosus GG; qPCR, quantitative polymerase chain reaction.

Overall, the results obtained from the qPCR were in agreement with the results from the culturing, although we observed a slight increase in the levels of the viable cell count of LGG when the supplemented milk sample was stored at room temperature for 6 hours, which was statistically significant (p = 0.046) (Supplementary Table S3). Moreover, the qPCR results for both probiotic strains were one log lower than the viable counts detected by the culturing method (Fig. 1). Slight differences in the concentrations, detected by the qPCR method, between the different time points might be because of the loss of DNA during extraction and purification procedures.

Discussion

In this study, we chose to use LGG and Bb12, as there is no clear indication on which probiotic strain is the most appropriate for administration to preterm infants and considering that both probiotic strains are generally considered safe for infant use.^11,12 To exert a beneficial effect on the host, probiotic concentrations between 1 × 10⁶ and 1 × 10⁸ CFU/mL in food have been suggested at the end of the storage period.¹³ For this reason, a concentration of 1 × 10⁸ CFU/mL for both probiotic strains was chosen. Initially, we expected that the viability of the added probiotics would decrease during storage under the examined conditions. This could be supported by the fact that LGG is unable to utilize lactose or intact casein and Bb12 is an anaerobic microorganism.¹⁴ In addition, we expected that their viability in human milk would be compromised since neither lactobacilli nor bifidobacteria grow well in cow's milk, although the growth rate could be strain dependent.^15,16 However, several studies suggest that the food matrix is an important factor that affects the viability of the added probiotics.¹⁷

Multiple factors may have an impact on the stability of the probiotics. For instance, the probable fermentation of human milk oligosaccharides by the added LGG and Bb12 could explain why both strains remain stable in this study.¹⁸ Regarding the viability of Bb12 in dairy products, the strain is able to utilize lactose and hydrolyze the milk proteins of bovine milk.^19,20

The storage conditions of our experiment did not have a significant impact on the probiotic counts examined by both culturing and genetic methods, and this finding could be explained by previous studies. For instance, Valik et al. have described that LGG can grow at a minimal temperature of 2.7°C in ultrahigh temperature-processed milk.²¹ In their study, Nighswonger et al. demonstrated that LGG remained stable during storage at 5°C to 7°C for 28 days in yoghurt and cultured buttermilk.²² In addition, Simpson et al. reported that freeze-dried Bb12 in skimmed milk remained viable during storage for 30 days at 4°C and presented moderate oxygen tolerance.²³ These observations are consistent with our findings. Furthermore, the strains isolated from the study matrix, namely S. epidermidis and S. warneri, did not have any negative impact on the viability of LGG and Bb12. However, the concentration of these bacteria was low (2 log CFU/mL) than the concentration of LGG or Bb12 (8 log CFU/mL). This may limit their interaction with the added probiotic strains.

Conclusions

In conclusion, the plate count and qPCR data indicate that both LGG and Bb12 are stable when supplemented in human milk and remain stable throughout the storage period. In addition, there was a tendency for LGG growth in the study samples when stored at room temperature for 6 hours, although culturing results were not statistically significant. Our results suggest that human milk could be a good matrix for probiotic supplementation. To our knowledge, this is the first study that has investigated the viability of LGG and Bb12 in human milk. Since the viability of added probiotics is critical for the delivery of the potential health effect, we believe that these findings can be of help for milk banks in hospital settings when human milk is supplemented with probiotics before its administration to the neonate. Finally, further studies in a larger number of samples are needed to evaluate the stability of added probiotics in human milk, as different storage conditions, processing procedures, probiotic strains, and different milk samples can present different results.

Footnotes

Disclosure Statement

No competing financial interests exist.

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