This study revealed that the AV, total FFAs, and caproic acid (C10:0 ) and lauric acid (C12:0) of human breastmilk increased with storage time, and that concentrations in the 30-day frozen milk samples were significantly higher than those in the 7-day frozen and fresh milk samples. These findings indicate that breastmilk’s rancid-flavor progressively developed with frozen-storage time.
Nevertheless, few studies have considered the flavor of human breastmilk; in contrast, the flavor quality of cow’s milk is strongly emphasized in the dairy industry since it affects customer acceptance and, in turn, the earnings of dairy producers . Therefore, to characterize the potential influence of the levels of FFAs, caproic acid, and lauric acid on the flavor of breastmilk, a comparison with the recommended sensory threshold of rancid flavor in dairy products was made. Early studies in dairy science have reported the relative levels of FFAs, caproic acid (C10:0), and lauric acid (C12:0) in terms of the sensory threshold for detecting the rancid flavor of milk [9, 25, 26].The detectable threshold usually means the minimum levels of a certain flavor compound perceived by at least 50% of the adult panelists ; accordingly, the FFAS levels in good-tasting milk should be lower than such thresholds .
Due to different analytical methods and panelist training, previous studies have reported considerable variations in the FFAs levels required for the detection threshold of the rancid flavor in milk, with a range from 1 to 3.62 mEq/100 fat . In response, the International Dairy Federation (1987) concluded that the FFAs levels for detecting the rancid-flavor threshold is normally between 1.5 to 2.0 mEq/100 fat for most adult consumers, with levels exceeding approximately 1.5 mmol/L reaching the unacceptable threshold [29, 30]. Specifically, the levels of caproic acid and lauric acid in milk required for reaching the rancid-flavor threshold were reported to be 7 and 8 ppm, respectively .
To compare with the aforementioned flavor thresholds, the values of AV and FFAs in this study were converted into the same units. The AV of fresh, 7-day frozen, and 30-day frozen milk samples in this study were approximately 2.55, 4.49, and 9.19 mEq/100g fat, respectively (unit conversion formula:1 mg KOH/g fat = 1 × 100 ÷ 56.1 mEq/100g fat; where 56.1 = molecular weight of KOH). In addition, the FFAs levels of the fresh, 7-day frozen, and 30-day frozen milk samples in this study were approximately 0.73, 1.32, and 2.63 mmolL−1, respectively (unit conversion formula:1mg/g fat = 1 × 28/260 mmolL−1; where 28 = g fat contained in per liter human breastmilk of this study, 260 = average molecular weight of FFAs estimated by the FFAs profile obtained in this study). These levels indicate that the flavor of fresh milk may already reach the rancid-flavor detection threshold, and that the flavor of frozen milk may easily exceed the unacceptable rancid-flavor threshold. When focusing on the representative compounds of rancid flavor, we found that caproic acid concentrations in fresh and frozen breastmilk seemed to be below the detection levels (approximate values: fresh: 1.4 ppm, 7-day frozen: 6.44 and 30-day frozen: 6.44 ppm; unit conversion formula : 1 mg/g fat = 1 × 28 ppm; where 28 = g fat contained in per liter human breastmilk of this study). In contrast, the lauric acid levels in the fresh and frozen breastmilk were much higher than the detection threshold for rancid flavor (approximate values: fresh: 12.04, 7-day frozen: 24.92, and 30-day frozen: 42.56 ppm).
Based on this comparison, this study found that the rancid flavor of the fresh breastmilk samples had already reached the rancid-flavor detection threshold for adults (2.55 mEq/100 fat); meanwhile, the FFAs levels in the 7-day (4.49 mEq/100 fat) samples far exceeded it and the 30-day frozen milk samples (2.63 ± 1.0 mmol/L) reached intolerance level. In addition, lauric acid, which has unclean and soapy flavor attributes , may be the main contributor to the rancid flavor of human breastmilk.
Our study revealed that human breastmilk seems to be susceptible to lipolysis, even when fresh, which accords with findings of other studies. For example, Lavine & Clark (1987) showed that the FFAs levels in fresh breastmilk were approximately 2.94 mEq/100g fat (0.23 ± 0.087 mg/ml) . Slutzah  found that the FFAs concentrations in fresh breastmilk analyzed within 2.4±1.2 hours of expression were approximately 4.48 mEq/100g fat (0.35 mg/ml). Another more recent study also indicated that human milk appears to be more susceptible to changes in flavor than bovine milk after frozen storage . Therefore, the principle of FFA levels being lower than the detection threshold of the rancid flavor in milk may not be appropriate for direct application in assessing human breastmilk flavor. Moreover, one point worth noting was that the sensory threshold of detecting the rancid flavor in milk was based on adult panelists. However, previous studies have shown that infants’ flavor sensitivity may differ from adults because even the protein hydrolysate formulas (PHF), which are perceived as extremely unpleasant for adults, are generally acceptable for infants younger than three months or those previously exposed to PHF . This implies that age and learning experience affect infants’ acceptance of food flavors . Therefore, despite rancid-milk flavor being described as foul and objectionable by adults , it may be acceptable by infants, especially those younger than three months old.
Based on our findings, we assume that the FFAs levels found in fresh breastmilk not only facilitate fat digestion for infants but also provide learning experiences for infants so as to facilitate becoming accustomed to the rancid flavor in frozen breastmilk. However, despite the lack of evidence indicating that milk lipolysis alters nutritive components in human breastmilk , it was noted that extreme lipolysis in breastmilk may increase the probability of infants refusing it, as demonstrated in Hung’s study . Therefore, we recommend that when infants refuse thawed milk, mothers could try to provide freshly expressed breastmilk or breastmilk frozen less than 7 days. Concurrently, the traditional principle of breastmilk use, “use the oldest milk in the refrigerator or freezer first” might be best implemented only when it has been frozen for less than 7 days to avoid infant feeding stress. Moreover, we recommend future studies explore methods for decreasing the rate of breastmilk lipolysis to prolong its acceptable flavor. Although pasteurizing has been proposed to reduce milk lipolysis by inactivating the lipase [9, 11], the appropriate procedure for women to pasteurize their milk safely requires further study.
As a preliminary study exploring the rancid-flavor compounds variations of human breastmilk under general frozen-storage conditions, several limitations are acknowledged. The first is that to include more women to participate this study was not easy because providing breastmilk for study may affect their infants’ feeding. Therefore, only 10 women with 30 milk samples were included in this study, which may affect the generalization of the findings. The second is that a direct sensory-perception test was not conducted in this study; accordingly, an assessment of infants’ acceptable thresholds of the rancid flavor in frozen breastmilk was not performed. Future studies could combine observations of infants feeding behaviors with quantitative analysis of flavor compounds to determine infants’ acceptable threshold for the rancid flavor of breastmilk. A third limitation is that there are many other reactions that can cause abnormal flavors in milk, e.g. lipid oxidation and proteolysis ; however, this study preliminarily focused on variations in the levels of breastmilk lipolysis during frozen storage. To extend this work, future studies could apply organoleptic testing through a trained panel and also analyze the objective compounds to explore the effects of lipid oxidation or proteolysis on breastmilk flavor under general storage conditions.