Lupins and Health Outcomes: A Systematic Literature Review

Lupins have a unique nutrient profile among legumes and may have beneficial health effects when included in the diet. The aim of this systematic review was to investigate the effects of lupin on a range of health outcome measures. Databases included MEDLINE, Embase and CINAHL, and focused on controlled intervention studies on healthy adults and those with chronic disease such as type 2 diabetes, cardiovascular disease and overweight. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocol was followed. Investigated intervention diets utilised whole lupin, lupin protein or lupin fibre, and outcomes were measured by markers of chronic disease, body weight and satiety. Quality assessment of results was performed using the Cochrane revised risk of bias tool. Overall, 21 studies with 998 participants were included: 12 using whole lupin, four used lupin protein and five lupin fibre. Beneficial changes were observed in 71% of studies that measured blood pressure, 83% measuring satiety and 64% measuring serum lipids. Unintended weight loss occurred in 25% of studies. Whole lupin demonstrated more consistent beneficial effects for satiety, glycaemic control and blood pressure than lupin protein or lupin fibre. Heterogeneity, low study numbers and a small participant base indicated further studies are required to strengthen current evidence particularly regarding the protein and dietary fibre components of lupin.


Introduction
Lupin (Lupinus) is a legume of the Fabaceae family that has formed part of the human diet since early civilisations. Legumes such as chickpeas, lentils, peas, beans and pulses continue to be a staple food in many parts of the world. Prized by consumers for their highly nutritious and inexpensive nature, they are becoming increasingly valued by producers for their ecological sustainability. With an increasing awareness of the benefits of regular legume consumption to human health, particularly among people living with chronic disease, lupin may be a useful addition to the food supply. Australia accounts for approximately 85% of the world's lupin production. It is grown predominantly in Western Australia, with some parts of New South Wales, Victoria and South Australia also under cultivation [1,2]. Lupin grows well in poor agricultural conditions, is pest-resistant and requires less water than many other food crops, therefore is ideally suited to Western Australia's climate and sandy soils. Moreover, it helps to improve soil fertility by the nitrogen fixing action of its rhizome, a characteristic shared with all legume plants [3]. The two most common varieties grown are the narrow-leafed lupin, also known as Australian sweet lupin or blue lupin (Lupinus angustifolius) and the white or albus lupin (Lupinus albus). Species of lupin cultivated globally include Lupinus mutabilis and Lupinus luteus or yellow lupin.
Macronutrient profiles of the various lupin species differ slightly. Compared with other legumes, Australian sweet lupin has one of the highest combinations of both protein this review if they met the following criteria: (a) controlled intervention trial of any duration and of parallel or cross-over design; (b) populations comprising any adults aged 18 years and above, with or without chronic disease, overweight or obesity; (c) dietary interventions in the form of whole lupin, such as dried, pickled and brined seeds, flakes, flour, crumb, meal, kibble and splits, or components of lupin such as protein and fibre; (d) assessed the effect of lupin consumption on biomarkers of chronic disease such as any related to diabetes, cardiovascular disease, hypertension, hypercholesteremia, hyperlipidaemia, cancer, inflammation, and oxidative stress, or assessed the effect of lupin consumption on anthropometric measurements and perceptions of satiety in relation to overweight and obesity and their association with chronic disease risk. The exclusion criteria applied to the study search were: (a) participants below 18 years of age; (b) assessments of single isolated proteins, peptides or alkaloids from lupin; (c) lupin intake as a supplement in capsule form; (d) non-English language studies; (e) publication dates before 1 January 2000.

Search Strategy
The following databases were searched: EMBASE (via Ovid) MEDLINE (via Ovid), CINHAHL (via EBSCO) from 1 January 2000 until 13 September 2021. Reference lists of eligible studies were scanned and searched manually on PubMed for additional studies.

Study Selection, Data Extraction, and Quality Assessment
Search results were imported into EndNote X9 ® referencing software (EndNote X9, Clarivate Analytics, Philadelphia, PA, USA) and duplicates were removed. Screening of studies was performed in two stages, first by title and abstract, then by full text. A data extraction form was created in Microsoft ® Excel ® spreadsheet (Microsoft 365 MSO Version 2109.14430.20306, Redmond, WA, USA) to include study citation, design, and duration; participant numbers and characteristics; intervention and control diet information; outcomes measured, and results obtained. The included studies were assessed for withinstudy risk of bias using the revised Cochrane risk-of-bias tool (RoB2) for randomised controlled trials [14]. Reviewer L.B. assessed studies to determine whether each study had low, some concerns, or high risk of bias. Areas of uncertainty were resolved in consultation with a second reviewer (S.G.). Assessment domains included risk of bias arising from the randomisation process, period and carryover effects, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported result [14].

Search Results and Study Selection
The search was conducted on 13 September 2021, returning a total of 157 records. One additional paper was identified from the reference lists of eligible studies. Following automated removal of duplicates by Endnote X9, 127 studies remained. Screening by title and abstract excluded 94 studies. Principal reasons for exclusion were publication type and study aim, interventions and measured outcomes beyond the scope of this review. A full-text review of the remaining 33 led to the exclusion of a further 12 studies. Reasons for exclusion were: duplications in other journals and/or non-English languages (n = 4), brief conference or workshop communications (n = 2), lupin intervention administration in capsule or supplement form (n = 2), investigation of lupin fractions such as alkaloids or single proteins (n = 3) and in vitro study protocol (n = 1). A total of 21 journal articles of controlled intervention studies met the inclusion criteria and were included in this qualitative review (Figure 1). Two studies generated three articles that reported on different sets of variables within each. These articles were treated as stand-alone studies and included in the final total.

Study Characteristics
Six of the included studies were randomised controlled trials (RCTs) of parallel design, the remaining 15 were cross-over studies, of which 11 were RCTs, while two were controlled, non-randomised, crossover studies. Research was based predominantly in Australia with smaller number of studies conducted in Germany, Italy and Ecuador. Table 1 lists study locations and the species of lupin under investigation. Five studies recruited healthy men and women [15][16][17][18][19], three recruited healthy men only [20][21][22], four studies involved men and women with type 2 diabetes [23][24][25][26], five with hypercholesterolaemia [27][28][29][30][31] and four involved people who were overweight or obese [32][33][34][35]. The number of completing participants ranged from n = 5 to n = 175 per study. Approximately 25% of the 998 participants across all studies were classified as healthy. Duration of study periods ranged from post-meal studies to 12 months. Categorisation of studies by form of lupin administered during treatment phases identified three distinct groups: whole lupin, lupin protein and lupin fibre. Study characteristics and outcomes of 12 whole lupin studies are summarised in Table 2, four lupin protein studies in Table 3, and five lupin fibre studies in Table 4. Measured outcomes were multiple and varied across studies, with p < 0.05 being declared as statistically significant.

Risk of Bias
Each study was assessed according to the criteria outlined in the revised Cochrane RoB2 tool for RCTs. All parallel studies had a low risk of bias. Most cross-over studies had a low risk of bias ( Figure 2); exceptions were one study with some concern for risk of bias in Domain1: Randomisation process, and in Domain 5: Selection of the reported result [25]. One other cross-over study had some concern in Domain 1 only [24].

Figure 2.
Within-study risk of bias assessment using the revised Cochrane risk-of-bias tool (RoB2) in 15 randomised (n = 11) and non-randomised (n = 2) controlled cross-over trials examining health outcomes of lupin consumption.

Range of Investigated Health Measurements and Their Outcomes
The five most investigated variables or groups of variables across all studies and the direction of lupin consumption effect are shown in Figure 3. These were: Figure 3. Percentage of total studies that reported differences between baseline and/or comparators (p < 0.05) by the five most investigated groups of health markers: serum lipids, glycaemic control, body weight, blood pressure and satiety, that had positive (desirable), negative (detrimental) or no effect on health outcomes. Serum lipids, which included any one or more of total cholesterol, LDL and HDL cholesterol, LDL:HDL ratio and triglycerides. Eleven studies measured serum lipids refs. [19,22,23,25,[27][28][29][30][31]34,35], of which 64% had one or more positive outcomes (i.e., showed statistically significant within-study difference(s) from baseline and/or control in a direction considered optimal for good health, depending on the health marker tested). Three studies did not report differences in serum lipids [19,23,34], while one study reported reduced HDL cholesterol with other lipids unchanged [35].

Whole Lupin
Seventy-five percent (n = 9) of all whole lupin studies (n = 12) reported a significant difference in at least one of the health markers under investigation, compared to baseline or to the control group. While most of the directions of change were desirable or positive outcomes for that variable, one study of overweight and obese participants reported decreased levels of HDL cholesterol, thereby reducing its potential heart-protective benefit, while other lipid markers did not change [35]. One study [25] reported increased serum glucose at 14 and 28 weeks and increased insulin levels at 28 weeks following a daily lupin snack in the first phase and two lupin snacks per day in the subsequent phase. Key health outcomes for whole lupin are shown in Figure 4a. All whole lupin studies (n = 4) that measured perceptions of satiety reported desirable changes [16][17][18]24], as well as 75% (n = 3) of studies that monitored blood pressure (n = 4) [25,33,35] and 67% (n = 6) of studies that measured glycaemic control (n = 10) [15][16][17]24,26,35]. Serum lipids moved to healthier levels in 25% (n = 1) of studies (n = 4) [25], as well as reporting decreased body weight measurements [25]. . Percentage of total studies categorised by (a) whole lupin, (b) lupin protein and (c) lupin fibre treatment that reported positive (potentially beneficial), negative (potentially detrimental) and no significant differences between baseline and/or comparators (p < 0.05) in the five most investigated groups of health markers: serum lipids, glycaemic control, body weight, blood pressure and satiety.

Lupin Protein
All lupin protein studies (n = 4) reported significant differences in at least one of the targeted health markers. All studies that measured serum lipids [27,28,30,31] and one study [30] of a total of two that measured blood pressure [30,31] reported significantly reduced levels (Figure 4b).

Lupin Fibre
All lupin fibre studies (n = 5) reported significant differences in at least one of the measured health markers. Two out of the three lupin fibre studies that measured serum lipids reported significant differences and positive health outcomes [22,29], as well as the one study that measured blood pressure [29] (Figure 4c). All three lupin fibre studies that monitored bowel function reported positive changes [19,21,29].

Discussion
This systematic review of the evidence for health outcomes from lupin consumption observed a range of results across many biological and anthropometric health markers that variously resulted in no effects, positive effects and negative effects in terms of optimum health. In the 21 studies that met the selection criteria, the strongest evidence related to a lowering effect on total cholesterol, LDL cholesterol and LDL cholesterol:HDL cholesterol ratio; reduction in systolic blood pressure, increased satiety and improvement in postprandial and glycaemic control. This supports the notion that lupin is equally and possibly more effective among all legumes in protecting long-term health. After categorising studies by form of lupin utilised in each intervention, this review noted potential relationships between lupins in their whole form and increased satiety perception, decreased blood pressure and improved glycaemic control, whereas lupin protein and lupin fibre demonstrated strongest positive results for blood pressure and serum lipids (though from a small study base).
These results correspond with the Kouris-Blazos et al. [8] review which concluded that sweet lupins may favourably effect blood pressure, blood lipids, insulin sensitivity and the gut microbiome. The Prusinski review [11] of white lupin concurred, stating that people who experience health conditions such as diabetes, hypertension, obesity, cardiovascular disease, hyperlipidaemia and colorectal cancer may benefit from the incorporation of this legume in the diet. However, research for the review centred on physiological properties of white lupin rather than on evidence for actual health outcomes. A 2017 review and meta-analyses investigating relationships between mortality and the intake of various food groups [36] found an inverse association between all-cause mortality and increased consumption of legumes, with no further dose response after 150 g/per day.

Whole Lupin
Nutrients in foods are metabolised in the human body according to the food matrix [37]. Categorisation of studies in this review by type of lupin administration, i.e., whole lupin, its protein and its fibre component, revealed greater health benefits were observed for the consumption of the whole food. Improved health outcomes were consistent for blood pressure [25,33,35], satiety [16][17][18]24] and glycaemic control markers [15][16][17]24,26,35] in whole lupin treatment studies, indicating benefits for reducing risk and managing symptoms of hypertension, cardiovascular disease, diabetes and obesity. Although it was noted that evidence for an increased satiety effect was present, evidence was less convincing for weight loss. None of the three whole lupin studies that measured body measurements detected a significant reduction in body weight [23,34,35]. Several reasons may account for this observation. Participants followed ad libitum diets that were not intended for weight loss, other lifestyle factors impacting weight, such as physical activity, were not monitored, and treatment duration may have been too short to demonstrate significant change. Hodgson et al. [34] proposed that if the observed trend in weight loss after four months was extended, a significant reduction of 2 kg could be expected within two years.
The study further proposed that while ad libitum diets that are high in protein and dietary fibre may result in loss of body weight, the amount of protein and fibre in whole lupin may be a factor. In addition, the mostly insoluble fibre present in lupin may not be as effective as isolated soluble fibre used in many dietary fibre weight loss studies. Nevertheless, the broad health benefits proposed by whole lupin consumption suggest a synergistic interplay of macro-and micronutrient components within the whole food matrix and their influence on multiple biological functions [37], leading to improved long-term health outcomes.

Lupin Protein and Fibre Components
Protein and dietary fibre components of lupin individually demonstrated consistent evidence for lipid lowering effects [22,[27][28][29][30][31]. These benefits were observed in hypercholesterolaemic participants, as well as in one of two studies that recruited healthy participants, yet whose average baseline total cholesterol was above 5 mmol/L [22]. Given that lipid levels did not change significantly in the lupin component study based on healthy men and women with average baseline total cholesterol below 5 mmol/L [19], this suggests lupin protein and fibre had moderating effects above this level. Evidence for blood pressure reduction from lupin component interventions with that of whole lupin does not conclusively favour its protein or fibre alone due to the paucity of studies. Serum lipids were measured by a similar number of studies across all three categories of lupin treatment. While results were variable, mostly positive effects were observed in diets that contained isolated lupin protein and lupin fibre. Substantially higher quantities of protein and fibre were provided in component trial protocols, compared with the amounts obtainable from the whole food in whole lupin studies. This may have contributed to the more consistently positive lipid outcomes in these lupin component studies. While the evidence for any health benefits from isolated lupin component consumption cannot be confirmed from so few studies, the addition of lupin protein to foods and beverages for maximising protein intake may be a useful alternative to soy and whey protein, particularly for consumers avoiding phytoestrogens and animal proteins. Similarly, lupin fibre is a gluten-free alternative for individuals with coeliac disease.

Dose Response
Indication of a dose response relationship between lupin and health outcomes was not identified due to multiple forms of lupin delivery and study methods among studies. However, one study [25] designed a dose response protocol comprising a doubled intake of whole Lupinus mutabilis during one intervention phase from 10 g to 20 g per day. While there was no change in glycaemic response markers between the two doses, blood pressure reduction was greatest after the increased dose phase.

Healthy vs. Unhealthy Participants Health Outcomes
Substantial evidence for health marker differences between healthy participants and those with type 2 diabetes, hypercholesterolaemia or who were overweight or obese, was unable to be determined because of the heterogeneity among studies. Having undertaken a sub-group analysis, however, one lupin fibre study [22] on generally healthy subjects found no significant effect on serum lipids among normocholesterolaemic participants (baseline total cholesterol < 5.5 mmol/L), while LDL cholesterol was significantly lowered among participants identified by study authors as 'mild to clinically hypercholesterolaemic' (baseline total cholesterol > 5.5 mmol/L). The investigation of whole Lupinus mutabilis consumption in type 2 diabetic subjects under conventional non-insulin medication found significantly reduced glycosylated haemoglobin (HbA1c) outcomes among a sub-group with less severe disease (HbA1c maintained at ≤ 8%), while no HbA1c effect was found in the remaining group that maintained HbA1C ≥ 8% and <10% [25]. Though changes in biomarkers may not be demonstrated consistently in healthy subjects, it can be supposed that lupin consumption offers protective benefits in hypercholesterolaemia and in wellcontrolled hyperglycaemia, if not in disease of greater severity.

Progression of Lupin and Health Outcomes Knowledge
Reflection on 16 years of lupin and health research revealed a progression from a focus on principal biomarkers for chronic disease, to a broader scope that encompassed other related health markers and possible biological mechanisms of effect. Analysis of faecal SCFA composition after lupin fibre consumption proposed that increased concentration and output of acetate and butyrate may have a protective effect on colorectal cancer risk [21]. A study on lupin fibre that observed a reduction in serum lipids in hypercholesterolaemic individuals proposed that increased bile acid excretion was not the result of bile acids binding to fibre, but a lower environmental pH from the fermentation of lupin fibre in the gut and the subsequent release of SCFAs [29]. Since the single study that focussed on lupin fibre and faecal gut bacteria was in 2006 [20], current interest and greater understanding of the gut microbiome warrants further investigation.

Strengths and Limitations
This systematic literature review was undertaken with the acknowledgement of several strengths and limitations. A major strength was the inclusion of high quality RCTs and non-randomised controlled studies that disclosed valid contextual reasons for nonrandomisation. Implementation of the revised Cochrane risk-of-bias tool facilitated recognition and acknowledgement of any limitations within studies. Limitations within this review relate to potential publication bias as only research published in English language journals was targeted. Relatively few studies met the selection criteria and the participant base was limited. Furthermore, the objectives, methods and analyses of the various studies lacked homogeneity, thus precluding a meta-analysis to be performed.

Future Directions
The subject of lupin consumption and health outcomes is a relatively new area of investigation, therefore more research is required to expand the evidence base. This should comprise multiple studies with similar aims, designs and protocols based on adequately sized population groups. Studies should identify the species of lupin and test all lupin forms in quantities that could feasibly be included in a normal diet, preferably in a dose response manner. This would allow for a more accurate assessment of the evidence overall for health benefits and optimum intake. In terms of health outcome measures, those for blood lipids, blood pressure and glycaemic control would be the most useful in identifying the unique nutritional and physiological properties of lupin. Furthermore, studies that involve concurrent investigations on healthy populations and those with different degrees of disease severity will inform whether lupin consumption may be more useful as a riskreduction strategy or in chronic disease minimisation.

Conclusions
This is the first systematic review to our knowledge to investigate the range of health outcomes and lupin consumption according to its mode of delivery, either as a whole food or the protein or fibre component. This review found divergent results in the effects of lupin consumption on many health marker outcomes, though greatest indications of benefit were apparent in improved satiety and reductions in blood pressure, and to a lesser extent in reductions in serum lipids and improved glycaemic control. More often, evidence was based on the whole lupin providing a broader range of health benefits than was observed in the smaller number of component studies. While the evidence for lupin's health benefits is promising, more substantial research would be required before health claims could be made. Nevertheless, its unique nutritional and physiological properties, particularly as a whole food, make it an ideal legume to include in a healthy diet.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article /10.3390/nu14020327/s1, Table S1: PICO (Population, Intervention, Comparator/Control, Outcome) framework to define the search strategy for the question: 'Is there an effect of human lupin consumption on health outcomes?', File S2: Search terms (MEDLINE).
Author Contributions: Conceptualization, L.B., S.G. and A.R.; methodology, L.B. and S.G.; formal analysis, L.B.; writing-original draft preparation, L.B.; writing-review and editing, L.B., S.G. and A.R.; supervision, S.G. and A.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding however was supported by the Grains & Legumes Nutrition Council, a not-for-profit charity organisation.