Rheological and microstructural characterisation of lotus seed milks and their glucono-δ-lactone induced acid-set milk gels: 1. Effect of protein content (2023)


Recently, there has been an increasing demand for milk alternative beverages derived from plant-based sources such as soy, oat, hemp, coconut, rice, and nuts, as animal milk substitutes. Reyes-Jurado et al. (2021) reported that the number of consumers who demand alternatives to bovine milk has significantly risen up to 61% since 2012. The expanding popularity of plant-based milk is attributed to several factors, including sustainability, healthy lifestyle, ethical concerns, lactose intolerance, dairy allergy, and animal welfare (Vogelsang-O’Dwyer, Zannini, & Arendt, 2021; Aydar, Tutuncu, & Ozcelik, 2020). Considering the effect of climate change and food security for the growing world population, more emphasis is being placed on plant-based diets (Poore & Nemecek, 2018; Willett et al., 2019). Generally, a plant-based milk is the extract of water-soluble compounds from plant materials with a milk-like appearance (Durand, Franks, & Hosken, 2003; Mäkinen, Uniacke-Lowe, O'Mahony, & Arendt, 2015). The most applied process for plant-based milk making includes separation of solid, product formulation, homogenization, heat treatment and packaging (Mäkinen, Wanhalinna, Zannini, & Arendt, 2016). Among plant-based milks, soy milk has been the most studied. Currently, soy milk-related products such as soy yoghurt, soy cheese and soy ice creams have been developed as an alternative to dairy-based products due to their nutritional benefits (Ningtyas, Tam, Bhandari, & Prakash, 2021). Soy protein provides a well-balanced amino acid composition and is a good source of nine essential amino acids (Nishinari, Fang, Guo, & Phillips, 2014). In addition to soy milk, other plant-based milks such as peanut, oat, almond, coconut, rice, and quinoa milks were successfully developed, while the global plant-based milk market keeps expanding (Jeske, Zannini, & Arendt, 2017). However, apart from soy protein, most plant proteins are often considered as incomplete due to a lack of some essential amino acids (Jäger et al., 2020). Vanga and Raghavan (2018) stated that there are various issues associated with current plant-based milk products, including low total number of calories, limited nutrient diversity, beany flavour and allergic reactions. These have motivated researchers to explore new plant sources as non-animal milk substitutes.

Lotus, Nelumbo nucifera Gaertn., is a perennial aquatic herb widely cultivated and consumed in Asia, Oceania and America (Liu et al., 2015). Most parts of the lotus exhibit excellent food and medicinal values (Zhang et al., 2015). Lotus seeds, the most prominent part, contain abundant functional ingredients, such as polyphenols, flavonols, procyanidins, alkaloids and polysaccharides (Liu et al., 2015; Yu et al., 2022). As the second largest component in lotus seed, lotus seed protein accounts for 19.85wt% on a dry weight basis (Zeng, Cai, Cai, Wang, & Li, 2013). Importantly, lotus seed protein reveals a high-quality amino acid composition, an FAO/WHO pattern of similar nutritional value to soy protein (Bangar, Dunno, Kumar, Mostafa, & Maqsood, 2022; Zeng et al., 2013). Zheng, Li, Zhang, Zheng, and Tian (2020) and Su et al. (2022) emphasized that lotus seed protein expresses excellent biological value with a high level of methionine and lysine and a lack of common allergens. Therefore, more attention has recently been paid to lotus seed proteins as a functional food ingredient (Zeng et al., 2013). Unlike soy protein, little information is available on the physicochemical and functional properties of lotus seed protein, which limits its application in the food industry (Jia et al., 2019).

Milk protein gelation is a significant step in cheese and yoghurt manufactures, which have great economic importance (Lucey, 2002). Over the last decade, there has been a growing interest in plant-based dairy products, including plant-based yoghurts and cheeses (Devnani, Ong, Kentish, & Gras, 2020). Recently, soy is the most popular plant-based source for yoghurt production because of its quantity, quality, and functional characteristics (Deng, 2021). Yang, Ren, Liu, Huo, and Li (2021) reported on the gelation mechanism of soy milk, which includes heat-induced protein denaturation and acid-induced protein coagulation. Heat-induced particle aggregates are an essential procedure for the formation of protein networks. Reducing the pH of heated soy milk to the isoelectric point results in protein aggregation (Ningtyas et al., 2021). Glucono-δ-lactone (GDL) is often used to mimic bacterial fermentation in model yoghurt systems and acid-induced gel (Vasbinder, van Mil, Bot, & de Kruif, 2001). It is an ester that slowly produces gluconic acid in water to decrease the pH, and is widely used to study the gelation of milk and soy proteins (Grygorczyk & Corredig, 2013; Kuipers, Alting, & Gruppen, 2007; Lucey, 2002). Grasso, Alonso-Miravalles, and O'Mahony (2020) pointed out that the main quality issues of plant-based yoghurts are appearance and texture properties, which are generally associated with phase separation. When plant-based milk is acidified, destabilization of the proteins leads to the formation of a non-continuous, weak gel, resulting in serum separation. Compared to dairy yoghurt, soy yoghurt exhibits a hard texture and lacks smooth sensations, making it hard to meet the requirements of consumers on smoothness, creaminess, soft texture and a less beany taste (Ningtyas et al., 2021). Mäkinen et al. (2015) investigated the physicochemical and acid gelation properties of UHT-treated commercial soy, oat, quinoa, rice and lactose-free bovine milk. Both soy and quinoa milk formed gels under the action of GDL with lower storage moduli compared with bovine milk gel. Further, the study showed that oat and rice milk did not gelify.

The physicochemical properties and acid gelation capability of protein, which play important roles in food systems, are not known for lotus seed milk. The current work was conducted to examine the primary structure, particle size distribution and viscosity of lotus seed milk. The effects of lotus seed milk concentration (5–20wt%) were also considered. In addition, the pH change during lotus seed milk gelation was monitored under the action of GDL. The properties of acid-induced lotus seed milk gels, including rheological properties, microstructure and syneresis, were determined. All the results on lotus seed milks and their acid-set gels were compared to 10wt% reconstituted skim milk and its acid-set gel as a control. To the best of our knowledge, this study is the first to report on the physicochemical and acid gelation properties of lotus seed milk.

Section snippets


Dried lotus seeds without seed coats and embryos were sold by Xiang Tan Lin Hong trading company Ltd. (Hunan, China) in 500g vacuumed packages without any additives. According to the label nutritional composition, every 100g of dried lotus seed contains 18.4g proteins, 1.2g fats and 65.5g carbohydrates. Low-heat skim milk powder (SMP) from Fonterra Ltd. (Hamilton, New Zealand). α-amylose (origin: Bacillus subtilis, 50 U/mg) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

Chemical composition and SDS-PAGE analysis

The chemical compositions of LSM powder and SMP are reported in Table 1. The moisture content of LSM powder (∼6.0%) is higher than that of SMP (∼3.6%) due to the different drying methods used; freeze-drying for LSM and spray drying for skim milk, and also to their difference in molecular contents and structures. The carbohydrate content, hydrolysed starch for LSM (∼52.9%) and lactose for SMP (∼51.7%) is similar for SMP and LSM powders, as was the lipid content, ∼0.70 and ∼0.69% for LSM powder


The LSM powder prepared in this study exhibited similar carbohydrate and lipid content to SMP, while its protein content was lower than that of SMP. Similarly to reconstituted skim milk powder (10wt%), LSM exhibited a negative redness (a*) appearance, and its yellowness (b*) at 10wt% was closer to that of 10wt% skim milk. However, the lightness (L*) of LSM, even at 20wt% concentration was darker compared to 10wt% skim milk. The particle size distribution of LSM was multimodal with two main

Author statement

Zhao Li: Methodology, Investigation, Data curation, Writing - original draft. Tingting Li: Investigation. Meng Zhao: Investigation. Bo Cui: Funding acquisition, Supervision. Yacine Hemar: Methodology, Data curation, Conceptualization, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


We would like to thank Dr. Hongshui Lu for his technical support with CLSM experiments. The research was supported by Key Research and Development Program of Shandong Province (No. 2021CXGC010808).

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