salivary amylase on starch experiment

Action of Salivary Amylase on Starch

To study the action of salivary amylase on starch solution.

The interaction between salivary amylase and starch constitutes a fundamental aspect of our digestive process, providing a glimpse into the complex biochemical mechanisms that enable our bodies to extract energy from the food we consume. Salivary amylase, an enzyme secreted by salivary glands, initiates the breakdown of complex starch molecules present in our diet. As food enters the mouth, the enzyme catalyses the hydrolysis of starch into simpler sugars like maltose. This enzymatic action marks the first step in carbohydrate digestion.

Experiment Procedure

To demonstrate the experiment on the action of salivary amylase on starch, we need to follow the given procedure:

• First of all, rinse your mouth with fresh water and collect saliva using a spatula/spoon.
• Then, filter saliva through a cotton swab.
• Now, take 1 mL of filtered saliva in a test tube and add 10 mL of distilled water to the test tube. Label it as “saliva solution.”
• Next, take 2 mL of 1% starch solution in 2 labelled test tubes (A and B).
• Add 1 mL diluted saliva to test tube B and shake well.
• We will not add anything to test tube A and keep it in control.
• After 5 minutes, take 5 drops from test tube A on a tile or a glass slide.
• Add 2 drops of 1% iodine solution in it, mix and observe colour.
• Place 5 drops from test tube B away from A’s mixture.
• Add 2 drops of 1% iodine solution to B’s drops, mix and observe.
• Repeat the iodine test after 5, 10, 15, and 20 minutes.

In conclusion, the experiment about “Action of Salivary Amylase on Starch” shows how enzymes in our bodies help break down food. By collecting saliva, diluting it, and mixing it with starch, it demonstrates how starch changes into simpler sugars. This change is important for getting energy from our food. Iodine solution is used to see this change, which makes the colour of the mixture different. These findings remind us how our bodies work with chemicals to stay alive. Understanding how salivary amylase acts on starch helps us see how our bodies make use of the food we eat.

FAQs on the Action of Salivary Amylase on Starch

Q.1 what is salivary amylase and its role in digestion.

Ans. Salivary amylase is an enzyme produced by the salivary glands, primarily in the mouth. Its main role is to initiate the digestion of complex carbohydrates, specifically starches, into simpler sugars.

Q.2 How does salivary amylase work on starch?

Ans. Salivary amylase breaks down the starch molecules into smaller fragments by catalysing the hydrolysis of the glycosidic bonds that link the glucose units in the starch molecule. This results in the production of maltose and other shorter carbohydrate chains.

Q.3 What factors can affect the activity of salivary amylase on starch?

Ans. The activity of salivary amylase can be affected by factors like pH, temperature, and the presence of inhibitors. An optimal pH level is necessary for its activity, and extreme temperatures or certain inhibitors might denature or inhibit the enzyme’s function.

Q.4 Why do we rinse the mouth with fresh water at the beginning of the experiment?

Ans. Rinsing the mouth with fresh water helps remove any residual food particles or substances that might interfere with the experiment. It ensures that only the collected saliva is being used in the experiment.

Q.5 What happens to the pH during salivary amylase action on starch?

Ans. Salivary amylase works optimally in a slightly acidic to neutral pH range, typically around pH 6.7. It starts the starch digestion process in the mouth, where the slightly acidic environment due to the presence of acids from foods and beverages helps activate the enzyme. 

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Salivary amylase and starch.

• + Create new collection

In this activity, students investigate the action of salivary amylase on starch present in cooked rice. Simple tests for starch and its digestion product, maltose, are applied.

By the end of this activity, students should be able to:

• use simple chemical tests to identify soluble starch and reducing sugars like glucose and maltose
• safely use their own salivary amylase
• explain in simple terms how the enzymatic digestion of starch occurs
• recognise the need for careful control of variables such as temperature and amount of reactant in activities of this type
• describe how high temperatures can inactivate enzymes like amylase.

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The article, Rate of digestion , looks at how surface area, temperature and pH all influence the rate of digestion of large food molecules. The action of salivary amylase on starch is used as an example.

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High Endogenous Salivary Amylase Activity Is Associated with Improved Glycemic Homeostasis following Starch Ingestion in Adults 1 , 2, 3

Abigail l. mandel.

4 Monell Chemical Senses Center, Philadelphia, PA; and

Paul A. S. Breslin

5 Department of Nutritional Sciences, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ

In the current study, we determined whether increased digestion of starch by high salivary amylase concentrations predicted postprandial blood glucose following starch ingestion. Healthy, nonobese individuals were prescreened for salivary amylase activity and classified as high (HA) or low amylase (LA) if their activity levels per minute fell 1 SD higher or lower than the group mean, respectively. Fasting HA ( n = 7) and LA ( n = 7) individuals participated in 2 sessions during which they ingested either a starch (experimental) or glucose solution (control) on separate days. Blood samples were collected before, during, and after the participants drank each solution. The samples were analyzed for plasma glucose and insulin concentrations as well as diploid AMY1 gene copy number. HA individuals had significantly more AMY1 gene copies within their genomes than did the LA individuals. We found that following starch ingestion, HA individuals had significantly lower postprandial blood glucose concentrations at 45, 60, and 75 min, as well as significantly lower AUC and peak blood glucose concentrations than the LA individuals. Plasma insulin concentrations in the HA group were significantly higher than baseline early in the testing session, whereas insulin concentrations in the LA group did not increase at this time. Following ingestion of the glucose solution, however, blood glucose and insulin concentrations did not differ between the groups. These observations are interpreted to suggest that HA individuals may be better adapted to ingest starches, whereas LA individuals may be at greater risk for insulin resistance and diabetes if chronically ingesting starch-rich diets.

Introduction

Saliva plays a vital role in maintaining the health of the oral cavity and gastrointestinal tract by aiding in lubrication, inhibiting potentially harmful microbes, and promoting oral tissue healing ( 1 ). Whether saliva also plays an important role in the digestion and metabolism of food is currently unknown. The presence of high concentrations of the enzyme α-amylase, however, has led to the hypothesis that saliva could be important for the digestion of complex carbohydrates ( 2 – 4 ).

Amylase is a digestive enzyme produced by the salivary glands and pancreas that cleaves the glycosidic linkages in starch molecules to produce smaller saccharides, such as maltotriose, maltose, and small amounts of glucose ( 5 ). Salivary amylase can account for up to 50% of total salivary protein in some individuals ( 6 ), whereas others produce barely detectable concentrations. Such substantial variation in amylase production is due to both environmental [e.g., stress (7)] and genetic factors, such as copy number variation (CNV) 6 in AMY1 , the gene that codes for salivary amylase. Copy number is positively correlated with salivary amylase concentrations ( 8 , 9 ). Individuals can carry anywhere from 1 to 15 diploid copies of the AMY1 gene in their genome.

Salivary amylase has been extensively studied since its discovery almost 200 y ago ( 10 ). Nevertheless, the fundamental question of whether the enzyme contributes to overall starch digestion and metabolism remains unanswered. Because food is only in the mouth for a few seconds, oral amylolytic “predigestion” is often assumed to be of minimal importance, particularly given the presence of pancreatic amylase within the gastrointestinal tract. However, there are hints that salivary amylase could be of practical and clinical importance. For example, we know that considerable starch hydrolysis occurs within seconds in the oral cavity ( 11 ) and can also continue after swallowing, because partially digested starch protects salivary amylase from acid inactivation ( 12 ). In vivo digestion studies demonstrate that delivery of starch directly into the small intestine, thereby skipping the oral digestion stage, results in substantially less starch digestion and glucose absorption ( 13 ). In addition, postprandial blood glucose concentrations following ingestion of starchy foods, such as rice and potatoes, are lower when the food is swallowed whole, rather than chewed first, mixed with saliva, and then swallowed ( 14 ).

Recent evidence suggests that populations who historically relied on starch for dietary energy have higher copy numbers of the AMY1 gene, with correspondingly higher concentrations of salivary amylase, than populations who consumed a high-protein diet ( 8 ). CNV of AMY1 may have evolved independently in diverse populations across the globe ( 8 ). This suggests that evolutionary nutritional pressures increased the number of AMY1 copies in select human populations, thereby facilitating the digestion and metabolism of starch. In contrast, pancreatic amylase, produced by the gene AMY2 , has not undergone similar genetic repetition ( 15 ) even though the vast majority of starch digestion occurs in the small intestine via pancreatic amylase ( 16 ). These observations collectively suggest that salivary amylase plays a critical role in the metabolism of complex carbohydrates.

Salivary amylase enables rapid cleavage of starch glycosidic linkages to produce smaller saccharides ( 5 ). We therefore surmised that individuals who produce more salivary amylase (group HA) would have faster and more substantial postprandial blood glucose responses following starch ingestion, due to more rapid starch breakdown, than individuals who produce less salivary amylase (group LA). We utilized a glucose solution, equivalent in energy to the starch solution, as a negative control. Because salivary amylase plays no role in glucose digestion, the 2 amylase groups should not differ in postprandial response.

Participants and Methods

Participant selection..

Adult volunteers were recruited from the surrounding area of Philadelphia and were of mixed ethnicity. Individuals initially underwent a screening by phone or email to assess eligibility; they were asked about height and weight, medical history, and cigarette use. Individuals with a BMI <25 kg/m 2 who reported no illness nor use of cigarettes or medications known to affect salivary flow were invited to participate further. Height and weight were verified in the laboratory and participants ( n = 48) were asked to provide a timed, stimulated saliva sample, which was analyzed for salivary amylase activity and flow rate. Participants were classified as either high or low amylase producers if their enzyme concentrations per minute (as calculated by salivary flow rate) fell 1 SD higher or lower than the group mean, respectively. Ten high amylase (HA) and 9 low amylase (LA) individuals (14 female, 5 male) participated in this study. Procedures were approved in accordance with the ethical standards of the Office of Regulatory Affairs at the University of Pennsylvania and all participants gave informed consent for participation on an approved form.

Experimental protocol.

Participants visited the laboratory for 2 separate morning sessions and had no food or beverages other than water since midnight of the previous night. The 2 sessions were at the same time on each day. Each individual participated in the experimental condition in which they consumed 50 g (10% solution) of a corn starch hydrolysate solution (M40; Grain Processing Corporation) and the control condition, in which they ingested 50 g (10% solution) of a glucose solution (Sigma Aldrich). The 2 solutions were equal in terms of energy provided. The starch hydrolysate was used in order to have a solution that did not noticeably differ in viscosity from the glucose solution. The glucose solution was prepared 24 h in advance to allow for complete mutarotation of the glucose tautomers. Participants were instructed to drink each solution at a constant rate over the course of 20 min and their rate of intake was monitored and timed. They were also instructed to swish every sip of solution around their mouth “like they would for mouthwash” for ~5 s before swallowing in order to fully mix the solution with saliva.

Before consuming the solutions, each individual gave a stimulated, whole saliva sample by chewing on a 4-cm square of parafilm for 90 s and expectorating into a 15-mL polypropylene tube. The tube was weighed before and after sample collection to calculate salivary flow rate (mL/min). The tube was vortexed, centrifuged at 2000 × g at 4°C for 10 min, and the saliva aliquoted and frozen at −80°C for future analysis.

For blood sampling, a butterfly needle was inserted into an antecubital vein and secured to the arm for the full duration of the study by a certified phlebotomist. All blood samples were collected into EDTA-coated tubes. Baseline blood samples were collected at −5 and 0 min. Following collection of the second baseline, the participant started to drink the carbohydrate solution. Blood samples were obtained every 3 min for the first 15 min and then every 15 min up to 2 h. The line was flushed with saline between samples to prevent clogging. Samples were immediately centrifuged and the plasma was aliquoted and frozen at −80°C.

For genotyping, ~5 mL of blood was collected from each individual into a tube coated with EDTA to prevent coagulation. The tubes were inverted gently 10 times and then frozen at −80°C for future use.

To assess dietary intake, participants completed a computerized Block 2005 FFQ (NutritionQuest). This validated questionnaire estimates the usual intake for a wide variety of foods and provides an analysis of overall carbohydrate, protein, and fat intakes. The data were also specifically analyzed for intake of starch-rich foods, including pasta, rice, bread, potatoes, etc. An intake frequency × quantity score was calculated for each food and the scores summed (FxQ Starchy Food Intake Score) to determine overall intake of starch-rich foods for each individual.

Biochemical measures.

Plasma glucose was analyzed in duplicate by a glucose oxidase method using a 2300 STAT Plus laboratory glucometer (YSI). Plasma insulin was analyzed in duplicate using a commercially available human insulin-specific RIA (HI-14K; Millipore). The assay was performed by the Diabetes Research Center of the University of Pennsylvania. Technicians were unaware of the conditions of the experiment.

Enzymatic activity assay for salivary amylase.

Upon thawing, saliva samples were centrifuged once more to ensure that solids were removed from suspension. Salivary amylase activity was determined using a chromogenic kinetic reaction assay kit (1–1902; Salimetrics), according to a previously described method ( 9 ).

qPCR for the AMY1 gene.

DNA was extracted from whole blood using the Gentra PureGene DNA extraction kit (Qiagen) and quantitated using a NanoDrop 2000C (Thermo Scientific). The diploid AMY1 gene copy number was determined using a Taqman Copy Number Assay for AMY1 (Assay ID Hs07226362_cn; Applied Biosystems),with a standard curve constructed from a reference DNA sample (NA18972; Coriell), as previously described ( 9 ).

Data analysis.

Participants were excluded from analysis if they exhibited resting blood glucose >6.1 mmol/L, resting insulin concentrations >140 pmol/L, or a peak blood glucose or insulin concentration more than twice the group mean on either study day.

Statistical analyses were performed using Statistica 9.0 software (Statsoft). Relationships between data sets were analyzed using the Pearson correlation coefficient. To determine between-group and between-treatment effects, incremental AUC was calculated for blood glucose and insulin as net change from baseline concentration (mean of 2 baseline samples) using the trapezoidal method. Glycemic index values were calculated as (starch AUC/glucose AUC) × 100. AUC values were compared using t tests. Peak blood glucose and insulin concentrations and other biological variables (age, BMI, salivary flow, amylase concentrations, and dietary intake) were also compared using t tests. CNV medians were compared using the nonparametric Mann-Whitney U test. Additionally, repeated-measures ANOVA was used to determine whether there were significant differences between the 2 groups or treatments during the blood sampling period. Repeated-measures ANOVA was also used to determine if there were significant differences within participants between baseline plasma glucose or insulin concentrations and subsequent measurements. For both tests, Tukey’s HSD post hoc pairwise analysis was used when significant interactions were found to determine which individual time points were significantly different from each other.

A 2-tailed P < 0.05 was considered significant. All results are presented as mean ± SE.

HA and LA groups.

Five individuals were removed from the analysis based on the exclusion criteria described in the “Methods.” This exclusion left 7 participants in the HA group and 7 in the LA group ( Table 1 ). The groups did not significantly differ in age or BMI. The salivary flow rate for the entire group (mean of 2 study days) was 1.58 ± 0.25 mL/min. The amylase concentration was 120 ± 24 kU/L and the amylase activity level was 202 ± 50 U/min. The HA group had greater salivary flow rate ( P < 0.05) and amylase levels in terms of both concentration ( P < 0.05) and rate ( P < 0.01). This result confirms that the groups were properly sorted by amylase concentrations. Enzyme concentrations were analyzed by amount and activity to ensure that the difference between the groups was not simply due to differences in salivary flow rate. There was a positive relationship between an individual’s amylase concentration (mean of the 2 study days) and their number of AMY1 gene copies ( r = 0.90; P < 0.0001) ( Supplemental Fig. 1 ). The HA group had more AMY1 gene copies than the LA group ( P < 0.05) ( Table 1 ).

Biological characteristics and dietary starch intake of healthy adult participants by salivary amylase activity 1

Plasma glucose and insulin responses following carbohydrate ingestion.

Following starch ingestion, plasma glucose concentrations differed over time between the 2 groups ( P < 0.01). Specifically, the HA group had lower postprandial glycemic responses at 45 ( P < 0.01), 60 ( P < 0.001), and 75 ( P < 0.01) min ( Fig. 1 ). The HA group also had lower incremental AUC (89 ± 21 vs. 244 ± 55 mmol/L · 120 min; P < 0.05) and peak blood glucose concentrations (9.56 ± 0.43 vs. 7.57 ± 0.35 mmol/L; P < 0.01) than the LA group. The 2 groups did not differ in their resting blood glucose concentrations (LA = 5.02 ± 0.13 mmol/L; HA = 4.99 ± 0.18 mmol/L). In both groups, blood glucose concentrations had risen above each group baseline within 15 min ( P < 0.05).

Postprandial plasma glucose concentrations in healthy, normal-weight adults by salivary amylase activity after ingestion of a 50-g starch solution. *Values are mean ± SE, n = 7. Asterisks indicate different from HA: * P < 0.01; ** P < 0.001. HA, high amylase group; LA, low amylase group.

Plasma insulin concentrations following starch ingestion did not significantly differ at any time point between the HA and LA groups when the curves were analyzed over the entire testing session ( Fig. 2A ). Because differences between low insulin concentrations during the preabsorptive period (before glucose absorption begins) may be masked by high concentrations later in the session, insulin concentrations for the first 9 min of the testing session were analyzed separately. The 2 groups differed during this period ( P < 0.05). The HA group had higher insulin concentrations at 9 min compared to their group baseline ( P < 0.01), whereas insulin concentrations for the LA group did not increase above their baseline at this time ( Fig. 2B ). The HA group also had higher insulin AUC values than the LA group for the 0 to 9-min period (144 ± 71.8 vs. −76.9 ± 20.9 pmol/L · 9 min; P < 0.01). There was a positive correlation between insulin production for the 0 to 9-min period (AUC) and the amount of oral amylase produced per minute ( r = 0.70; P < 0.01) ( Supplemental Fig. 2 ).

Postprandial plasma insulin concentrations in normal-weight individuals by salivary amylase activity after consumption of a 50-g starch solution over the entire testing session ( A ) and during the preabsorptive period (0–9 min) ( B ). For B , each group was compared against their own baseline. The data are portrayed as change from baseline (Δ) in order to highlight the differences between the groups. Values are mean ± SE, n = 7. *Significantly different from baseline, P < 0.01. HA, high amylase group; LA, low amylase group.

Glycemic responses following ingestion of the control glucose load did not differ between the amylase groups at any time point ( Fig. 3A ), nor were there differences in AUC or peak blood glucose concentrations (data not shown). Furthermore, plasma insulin response did not differ between the 2 groups either overall or in the first 9 min ( Fig. 3B ). Notably, both groups had insulin concentrations higher than baseline within 9 min, indicating that both groups were capable of preabsorptive insulin responses to the glucose solution (HA, P < 0.01; LA, P < 0.05) ( Fig. 3C ).

Postprandial plasma glucose ( A ) and insulin ( B , C ) concentrations after consumption of a 50-g glucose solution. For C , each group was compared against their own baseline. The data are portrayed as change from baseline (Δ). Values are mean ± SE, n = 7. Asterisks indicate significantly different from baseline: * P < 0.05, ** P < 0.01. HA, high amylase group; LA, low amylase group.

Within-participant comparisons.

Blood glucose concentrations following starch and glucose ingestion did not differ within each group ( Supplemental Fig. 3 ). However, the LA group had a larger AUC following starch ingestion (244 ± 55 mmol/L · 120 min) compared to the glucose load condition (152 ± 48 mmol/L · 120 min) (not shown; P < 0.005). Accordingly, the LA group (111 ± 7) had a significantly higher glycemic index for the starch solution than the HA group (94 ± 3) ( P < 0.05).

Dietary intake of carbohydrates.

Analysis of the FFQ data for each participant demonstrated that the groups did not significantly differ in terms of overall carbohydrate intake or intake of high-starch foods ( Table 1 ).

In the current study, we tested whether high salivary amylase concentrations altered blood glucose responses following starch ingestion. We hypothesized that because starch is cleaved into simple sugars by salivary amylase, people possessing high salivary amylase concentrations (group HA) might thus be expected to have higher postprandial blood glucose following starch ingestion relative to participants with lower salivary amylase concentrations (group LA). Instead, we found the opposite occurred: compared with LA individuals, HA individuals had significantly lower postprandial blood glucose responses following starch ingestion. This difference was apparently mediated by the increased plasma insulin concentrations in the HA group observed early in the testing session. Nevertheless, both groups had similar plasma glucose and insulin responses following glucose ingestion. Thus, it is unlikely that group differences were due to innate differences either in their ability to produce insulin or in their capacity for insulin-mediated glucose disposal.

Plasma glucose concentrations following starch ingestion did not begin to rise in either group until 15 min into the session and, therefore, the early insulin release described above can be termed preabsorptive (occurring during the preintestinal absorption period). It has been known since the work of Ivan Pavlov more than 100 y ago that the flavor of food or food ingestion can stimulate anticipatory digestive and metabolic responses, prior to nutrient absorption, that result in the increased secretion of saliva ( 17 ), gastric acid ( 17 , 18 ), and pancreatic secretions ( 17 , 19 ). Such responses presumably prepare the digestive system to digest food, as well as absorb and metabolize nutrients ( 20 ). This strategy increases the efficiency of digestion and metabolism and also enables better maintenance of homeostasis ( 20 , 21 ).

Preabsorptive insulin release, also known as cephalic phase insulin release (PIR) is one such anticipatory response to eating ( 22 ). Though it is a relatively minor component of total insulin secretion, PIR is an extremely important determinant of overall glucose tolerance ( 23 ). Studies in both laboratory animals and humans have demonstrated that loss of this response leads to impaired glucose tolerance ( 24 , 25 ). For example, i.g. administration of glucose in rats, which bypasses the oral cavity, leads to delayed insulin release and much higher blood glucose concentrations than when the same amount of glucose is orally ingested ( 24 ). Similarly, the LA group in the current study did not exhibit PIR in response to starch and consequently had a higher glycemic response. After ingesting the glucose solution, however, both groups exhibited PIR, which indicates that such a response can be elicited in the LA group.

Though the specific process by which salivary amylase stimulates PIR and affects glucose homeostasis remains unclear, we offer several possibilities. One possibility is that the production of glucose and/or maltose through amylolytic activity in the oral cavity signals the body to prepare for incoming starch and the ensuing glucose. The sugars would bind lingual T1R2-T1R3 sweet taste receptors ( 26 ) and/or glucose transporters in taste receptor cells ( 27 ). Because the amount of glucose produced by salivary amylase is too low to be consciously tasted and maltose is only weakly sweet tasting, the stimulation of these taste receptors would not be expected to activate perceptible sweet taste ( 28 ). Second, the mechanism may also involve binding of short-chain oligosaccharides by the putative polysaccharide receptor, hypothesized to enable identification of starch-rich foods ( 29 ). Finally, it is also possible that hormones or incretins (e.g., glucagon-like peptide-1) are peripherally released by lingual taste cells into the blood stream in response to carbohydrates, stimulating insulin release from the pancreas during the PIR period.

With the advent of agriculture and the domestication of cereals such as barley, wheat, maize, and rice, the reliance on starches for dietary energy dramatically increased in many regions of the world. Evolutionarily, increased AMY1 copy number and salivary amylase concentrations would provide a considerable nutritional advantage following this dietary change. Efficient starch digestion would have been of immense benefit, providing rapid replenishment of blood glucose following periods of intense energy expenditure, such as during farming, active hunting, or episodes of lower gastrointestinal malaise or toxicosis.

In today’s society, starches contribute over one-half of the total carbohydrate energy consumed in the US ( 30 ). More than 85% of these starches are highly processed and refined ( 31 ), similar to the starch solution in the current study. “Dietary globalization” has led to widespread availability of these highly refined, starch-rich foods and therefore it is perhaps not surprising that we did not find any differences in carbohydrate intake between the HA and LA groups. However, although these 2 groups eat similar foods, our data suggest that they experience different glycemic responses to them. This has potential implications for the calculation of glycemic indices for starch-rich foods because the current method does not take into account individual differences in starch digestion. It may, therefore, be necessary to calculate different glycemic indices for individuals with different amylase concentrations.

The imbalance between genetic background and evolutionary optimized diet may also have potential implications for the development of noninsulin dependent diabetes and obesity. The reasons why some individuals develop these conditions while others do not are not currently understood. In light of our current findings, we suggest that AMY1 gene copy number may play a role in the development of insulin resistance and diabetes. Both high and low amylase individuals in this study were young and healthy, with a mean BMI <22 kg/m 2 , yet the groups had different glycemic responses following starch ingestion. Although overall insulin concentrations did not differ between the groups, it is possible that chronic high blood glucose concentrations induced by high starch intake may elicit a number of hormonal, receptor, and physiological changes that will eventually result in the development of insulin resistance and diabetes. We suggest that it may, therefore, be useful to begin testing individuals for low AMY1 gene copy number and salivary amylase concentrations to help assess risk for these conditions.

One potential limitation of this study was our use of a liquid starch hydrolysate solution for our experimental condition. A previous study involving the mastication of more complex starch-rich foods found that blood glucose concentrations were higher if the food was first chewed and then swallowed rather than swallowed whole ( 14 ). It will be necessary to verify our findings with future studies of more complex starch-rich foods.

To our knowledge, this is the first report demonstrating that salivary amylase interacts with certain ingested complex carbohydrates to affect insulin and blood glucose concentrations. This research provides a possible explanation for the benefits of the oral predigestion of starch as well as the benefits of high AMY1 gene copy number and salivary amylase production. Our results indicate that individual differences in salivary amylase may considerably contribute to overall nutritional status.

Acknowledgments

The authors thank Karen Teff and Louise Slade for their assistance with experimental design, Suzie Alarcon for analysis of AMY1 gene copy number, Huong-Lan Nguyen for assistance with blood glucose analyses, Anthony King for phlebotomy assistance, Gary Beauchamp for comments on the manuscript, and the Coriell Cell Repository for the reference DNA sample used to quantify copy number variants. A.L.M. conducted the study and analyzed the data; and both authors designed the research, provided reagents/materials, wrote the paper, and had responsibility for the final content. Both authors read and approved the final manuscript.

1 Supported by a Ruth Kirschstein Individual Postdoctoral National Research Service Award (DK084727) to A.L.M. and an RO1 (DC02995) to P.A.S.B.

3 Supplemental Figures 1–3 are available from the “Online Supporting Material” link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org .

6 Abbreviations used: CNV, copy number variation; HA, high amylase group; LA, low amylase group; PIR, preabsorptive insulin release.

Literature Cited

Study the Effect of Temperature on Salivary Amylase Activity

• First Online: 28 February 2020

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• Aakanchha Jain 4 ,
• Richa Jain 5 &
• Sourabh Jain 6  

Part of the book series: Springer Protocols Handbooks ((SPH))

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Enzymes are proteinaceous in nature and catalyze chemical reaction in biochemistry. Enzymes are responsible for speeding up reaction and mostly synthesized in living cells. A study of enzymatic hydrolysis of starch will give knowledge about specific reactions of enzymes. There are several factors like temperature and pH that affect the reaction. At higher temperature the enzymes are denatured, while at lower temperature, the enzymes are deactivated, so this takes more time at low and high temperature to digest the starch. At optimum temperature (32–37 °C), the enzyme is active and therefore consumes less time for starch digestion.

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Bhagyoday Tirth Pharmacy College, Sagar, Madhya Pradesh, India

Aakanchha Jain

Centre for Scientific Research and Development, People’s University, Bhopal, Madhya Pradesh, India

Sagar Institute of Pharmaceutical Sciences, Sagar, Madhya Pradesh, India

Sourabh Jain

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Jain, A., Jain, R., Jain, S. (2020). Study the Effect of Temperature on Salivary Amylase Activity. In: Basic Techniques in Biochemistry, Microbiology and Molecular Biology. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9861-6_53

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DOI : https://doi.org/10.1007/978-1-4939-9861-6_53

Published : 28 February 2020

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Oral Digestion and Perception of Starch: Effects of Cooking, Tasting Time, and Salivary α-Amylase Activity

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Trina J Lapis, Michael H Penner, Amy S Balto, Juyun Lim, Oral Digestion and Perception of Starch: Effects of Cooking, Tasting Time, and Salivary α-Amylase Activity, Chemical Senses , Volume 42, Issue 8, October 2017, Pages 635–645, https://doi.org/10.1093/chemse/bjx042

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Since starch is a significant part of human diet, its oral detection would be highly beneficial. This study was designed to determine whether starch or its degradation products can be tasted and what factors influence its perception. Subjects were asked 1) to taste 8% raw and cooked starch samples for 5, 15, and 35 s and rate perceived intensities of sweetness and “other” taste (i.e., other than sweet), 2) to donate saliva to obtain salivary flow rate (mg/s) and salivary α-amylase activity (per mg saliva), and 3) to fill out a carbohydrate consumption survey. Subsequently, in vitro hydrolysis of starch was performed; saliva was collected from 5 subjects with low and high amylase activities and reacted with 8% raw and cooked starch at 2, 15, and 30 s. Hydrolysis products were then quantified using a High performance liquid chromatography. The results showed cooking increased the digestibility of starch such that the amount of hydrolysis products increased with reaction time. However, cooking did not influence taste ratings, nor were they influenced by tasting time. Subjects’ salivary amylase activities were associated with the efficacy of their saliva to degrade starch, in particular cooked starch, and thus the amount of maltooligosaccharide products generated. Effective α-amylase activity [i.e. α-amylase activity (per mg saliva) × salivary flow rate (mg/s)] and carbohydrate consumption score (i.e. consumption frequency × number of servings) were also independently associated with sensory taste ratings. Human perception of starch is undoubtedly complex as shown in this study; the data herein point to the potential roles of salivary α-amylase activity and carbohydrate consumption in the perception of cooked starch.

Carbohydrates are the most abundant and diverse class of organic compounds occurring in nature ( Izydorczyk 2005 ). Even considering only those readily utilized and metabolized in human body, carbohydrates encompass a wide range of molecules and can be classified into 3 groups: mono- and disaccharides (low molecular weight), oligosaccharides (intermediate molecular weight) and polysaccharides (high molecular weight). Although all 3 classes of saccharides are present in natural food products (i.e. fruits, vegetables, edible roots, and grains), research on human gustatory responses to carbohydrates has focused almost exclusively on mono- and disaccharides (i.e. simple sugars). It is perhaps due to the long standing assumption that simple sugars are the only class of carbohydrates that humans can taste ( Jacobs 1962 ; Feigin et al. 1987 ; Ramirez 1991a ).

In contrast, there has been considerable amount of research on animals’ gustatory responses to complex carbohydrates. Studies have shown that rodents ( Sclafani 1987 ) and some non-human primates ( Laska et al. 2001 ) can taste starch-derived glucose oligomer/polymer mixtures (e.g. maltodextrin, polycose). More recently, our lab demonstrated that human subjects can perceive maltodextrins, commercially available mixed glucose oligomer/polymer preparations, in the absence of olfactory cues ( Lapis et al. 2014 ). In that study, it was shown that responsiveness to maltodextrin samples were highly correlated with one another but not necessarily with glucose and sucrose, whereas the responsiveness to glucose and sucrose were significantly correlated. These findings suggest that humans can sense glucose oligomers and/or polymers and that such responsiveness is independent of that for simple sugars. In order to better understand the target substrates that humans can taste, we recently developed an approach to decrease the degree of polymerization (DP) heterogeneity of maltodextin preparations and the samples with average DP 7, DP 14, and DP 44 were produced ( Balto et al. 2016 ). A psychophysical study revealed that human subjects could discriminate glucose oligomers (i.e. average DP 7 and 14) from water blanks in the absence of olfactory and textural cues while salivary α-amylase was inhibited. The same subjects, however, could not discriminate glucose polymers (i.e. average DP 44) under the same condition ( Lapis et al. 2016 ). A focus group included in the study described the taste quality of glucose oligomers as “starchy,” like root vegetables, corn, bread, and pasta, not sweet ( Lapis et al. 2016 ).

Starches account for 60–70% of all calories consumed by humans in modern environment ( Robyt 2008 ). Starch can be found in various botanical sources such as cereals and root vegetables. It consists of 2 types of glucose polymers; amylose, about 1000 glucose units linked by linear α-1,4 glucosidic linkages, and amylopectin, an average of ~100 000 glucose units linked predominantly by linear α-1, 4 glucosidic linkages along with branched α-1,6 glucosidic linkages ( Wang et al. 1998 ; Robyt 2008 ). Taste perception of starch was thought to be unlikely, since starch is stored in plants as water insoluble granules, which are not expected to interact with taste receptors ( Birch 1987 ; Ramirez 1991a ; Wang et al. 1998 ). However, when aqueous starch suspensions are heated at or above their gelatinization temperature (i.e. 61–99°C depending on the botanical source) ( Biliaderis et al. 1980 ; Robyt 2008 ), the starch granules swell, lose their crystalline character, and starch molecules begin to leach into solution ( Robyt 2008 ). Solubilized starch is still likely to be too bulky to be the ligand for a taste receptor ( Birch 1987 ; Ramirez 1991a ), but the concurrent actions of mastication and enzymatic hydrolysis during oral digestion provides a degradation environment that generates lower molecular weight glucose oligomers. Salivary α-amylase, an endo-acting enzyme, specifically catalyzes the hydrolysis of α-1,4 glucosidic linkages in the interior of the polymeric starch chains to produce shorter chain saccharides including maltose, maltotriose, and larger glucose oligomers ( Robyt and French 1970 ; Jacobsen et al. 1972 ; Robyt 2008 ).

Various factors affect rates of starch saccharification. Acidic environments (e.g. acidic drinks) slow starch hydrolysis because they retard salivary amylase activity ( Hanson et al. 2012a , 2012b ). On the other hand, cooking has been shown to increase the rate of oral starch hydrolysis due to gelatinization and solubilization, both of which make the starch more enzyme accessible ( Snow and O’Dea 1981 ). In addition, it was shown that starch breakdown occurred within a short tasting time period (e.g. 10–20 s) and as the reaction time increased, the extent of starch hydrolysis increased ( Snow and O’Dea 1981 ; de Wijk et al. 2004 ; Ferry et al. 2004 ; Hanson et al. 2012a , 2012b ). Genetic variation and diet also appear to relate to individual salivary α-amylase activities. Copy number of the salivary α-amylase gene (i.e. AMY1 ) was found to be positively correlated both with salivary α-amylase protein levels ( Mandel et al. 2010 ) and with diets high in starch ( Perry et al. 2007 ). Individuals with high salivary amylase levels were found to hydrolyze starch solutions faster and more significantly than those with low salivary amylase levels based on time-intensity ratings of viscosity, which track the digestion of starch during oral manipulation ( Mandel et al. 2010 ). These studies provide insights into the initial digestion of starch in the oral cavity ( Hoebler et al. 1998 ; de Wijk et al. 2004 ; Ferry et al. 2004 ; Hanson et al. 2012b ) and the associated perceptual changes of oral viscosity or thickness of foods ( de Wijk et al. 2004 ; Mandel et al. 2010 ). However, previous studies have not examined the effects of salivary amylase and related factors on taste perception of starch.

The current study was thus designed to investigate taste perception of starch and the factors that influence its perception. The factors considered were method of sample preparation (i.e. cooking), length of tasting time, individual differences in salivary α-amylase activities and extents of complex carbohydrate consumption. Extra oral digestion of starch was also investigated through an in vitro salivary α-amylase hydrolysis. It was hypothesized 1) that starch itself cannot be tasted because of its insolubility and bulky structure; 2) that cooking would gelatinize/solubilize starch making it more susceptible to salivary α-amylase and hence leading to the concomitant production of starch hydrolysis products; 3) that levels of salivary α-amylase activity influence oral digestion and hence the taste perception of starch; and 4) that the amount of daily carbohydrate intake correlates with one’s ability to perceive starch.

Part I: taste perception of starch

A total of 60 subjects (40 F, 20 M) between 18 and 44 years of age (mean = 23 years old) were recruited from the Oregon State University campus and surrounding area. Individuals who were interested in participating in a taste perception study were asked to fill out a screening questionnaire, which consists of questions about general health. Subject inclusion criteria were individuals who are: 1) non-smokers; 2) not pregnant; 3) not diabetic; 4) not taking any prescription medication; 5) free from taste deficit or other oral disorders; and 6) without a history of food allergy. Respondents who met all of the above criteria were invited to participate in the study. In order to prevent deviation from normal α-amylase activity, subjects were asked to comply with the following restrictions prior to their test sessions: 1) no dental work within 48 h; 2) no alcohol consumption within 12 h; 3) no consumption of foods and beverages that are acidic or caffeinated, and/or contain dairy within 4 h; 4) no consumption of food or beverage of any kind except water within 1 h; 5) no use of any menthol-containing products (e.g. toothpaste, mouthwash, chewing gums) within 1 h; and 6) no physically demanding activity on the morning of their scheduled sessions ( Chatterton et al. 1996 ; Rohleder and Nater 2009 ). The experimental protocol was approved by the Oregon State University Institutional Review Board and complies with the Declaration of Helsinki for Medical Research. Subjects gave written informed consent and were paid to participate.

A total of 3 test stimuli, prepared as aqueous solutions (% w/v), were used in the experiment: 6% glucose (Sigma-Aldrich) and 8% raw and cooked corn starch samples (73% amylopectin, 27% amylose; S4126 Sigma-Aldrich). Aqueous solutions of 8% glucose and 8% raw and cooked starch were also used as practice stimuli. The cooked starch sample was prepared by heating a starch suspension on a temperature-controlled magnetic stir plate at 90°C for 20 min with continuous stirring using a magnetic stir bar. The concentration of starch was chosen to use the maximum possible amount that can be incorporated into the solution without incurring difficulty while stirring the starch samples. Concentrations higher than 8% rendered the starch too viscous while cooking and too pasty upon cooling. Glucose solution, a relatively non-viscogenic stimulus, was included as a taste control and its concentration (6%) was determined during pilot tests. The concentration was chosen because it elicits a weak sweet sensation that can be clearly differentiated from the taste of starch. Glucose solution was prepared at least 12 h before the test session to allow for complete mutarotation of glucose tautomers ( Pangborn and Gee 1961 ) and was stored at 4−6°C. Raw and cooked starch samples were prepared fresh daily. All stimuli were prepared with deionized water and were allowed to come to room temperature (21–23°C) before being provided to the subjects.

Experimental protocol

Each subject participated in one experimental session, which was divided into 3 parts: psychophysical study, saliva collection, and completion of survey. Since salivary α-amylase levels usually follow a diurnal pattern ( Nater et al. 2007 ), all subjects were tested in the morning, from 9 to 11 AM. The testing was conducted on a one-on-one basis in a psychophysics laboratory.

Psychophysical procedure

Data were collected using the general version of the Labeled Magnitude Scale (gLMS) ( Green et al. 1993 , 1996 ; Bartoshuk et al. 2003 ). The gLMS was chosen to capture perceived intensities of the samples along with their corresponding semantic labels and also to make valid comparisons across individuals and groups. Prior to psychophysics data collection, subjects were verbally instructed on how to use the general version of the Labeled Magnitude Scale (gLMS). The scale was displayed on a computer monitor and subjects used a mouse to move a cursor along the scale to make their ratings. After the experimenter explained the unique structure of the scale in detail, the subjects rated a list of 15 remembered or imagined sensations (e.g. the sweetness of cotton candy, the heat of drinking boiling hot tea, the weight of a feather in the hand) to practice using the gLMS in the broad context of sensations encountered in everyday life.

In order for the subjects to become acquainted with the task to be performed and the different types of stimuli, subjects were given practice ratings before proceeding to actual taste intensity ratings. During practice, taste quality descriptors were first generated from each subject. Subjects were instructed to actively taste 1 mL of practice stimuli (8% raw and cooked starch, 8% glucose) by making gentle smacking motions between the tongue and the roof of the mouth for 15 s, expectorate and describe the taste qualities perceived from the stimuli. Active tasting was encouraged to allow for more efficient incorporation of saliva into the stimuli and also to mimic a more natural eating condition. To eliminate any olfactory input from the test stimuli, subjects were asked to wear nose clips when tasting the stimuli, which were found to be effective in rendering odorous compounds odorless ( Hettinger et al. 1990 ). Since subjects wore nose clips, which posed difficulty in breathing they were told to breathe by inhaling and exhaling while they open their mouths halfway (after 7 s) during the tasting period. They were not allowed to remove the nose clips until after they had provided taste quality descriptors and also were prompted to breathe at the 7-s time point. Once quality descriptors were obtained, subjects were asked to rate the perceived intensities of sweetness and “other” taste after actively tasting 1 mL of 8% cooked starch for 15 s. “Other” taste was defined as any taste quality other than sweetness, which included the quality descriptors that the subject described for starch (e.g., “cracker-”, “flour-”, “rice-”, “tapioca-”, and “starchy”). In the rare case that subjects could not provide specific descriptors, they were told that “other” taste can include qualities such as “bread-”, “cracker-”, “cereal-”, or “rice-like”. After indicating their ratings, subjects were also asked to notice the changes in the texture of test samples and verbally describe mouthfeel (e.g. gel-, pudding-like, thick) perceived from the stimuli. This encouraged the subjects to recognize the possible changes of test samples (e.g. from thick to thin in the case of cooked starch) and to consider mouthfeel separately from taste and thus to help prevent dumping of mouthfeel into taste ratings ( Lawless and Clark 1992 ; Clark and Lawless 1994 ).

Once the training was completed, the actual testing proceeded following the tasting procedure mentioned above. During the actual test, subjects tasted the test stimuli for 3 different time periods: 5, 15, and 35 s while wearing nose clips. Since the nose clips made breathing impossible through the nose, subjects were asked to breathe through their mouth once during the 15-s tasting time period (after 7 s) and 3 times during the 35-s tasting time period (after 9, 18, and 27 s). Again, subjects were prompted to breath at the designated time points. Each time period served as a tasting block. The orders of tasting block and stimuli presentation within each tasting block were randomized and counter-balanced across subjects. A 3-min break was provided between each tasting block. All samples were directly put in the subjects’ mouth one by one by the experimenter using mini spoons. This was done to avoid spills and to ensure convenient and consistent delivery of the stimuli into the subjects’ mouths. Subjects rinsed at least 3 times with 37°C deionized water during a 1-min inter-stimuli interval.

Saliva collection

Whole, stimulated saliva was collected from each subject. Subjects were asked to chew on a 3-cm plastic drinking straw to the beat of a metronome (80 beats/min) for 60 s and then expectorate their saliva into pre-weighed polypropylene tubes. This protocol is intended to provide an estimate of saliva production rates amongst individuals, although it is recognized that this method of stimulating saliva flow is not directly equivalent to the lip smacking used during perception testing. It is also recognized that salivary amylase activities per mg saliva may not be constant throughout saliva flow ( Schneyer and Schneyer 1960 ) and thus the reported amylase activity values for which total saliva produced was taken into account must be considered to be correlates of the average amounts of amylase activity per mg saliva. The tubes were weighed after the saliva collection to calculate the rate of salivary flow (mg/s). Saliva samples were stored at 4–6°C until assayed for salivary amylase activity, which was conducted in the afternoon of the same day. The salivary α-amylase assay was conducted following the procedure described in Lapis et al. (2014) . The colorimetric assay is based on the enzyme-catalyzed liberation of soluble, colored starch fragments from an insoluble, dye-labeled, cross-linked starch substrate. The assay contained a standardized amount of CaCl 2 to eliminate effects due to variation in the concentration of these ions. One salivary α-amylase activity unit (SAU) was defined as amount of saliva that changes the absorbance of the test solution by one absorbance unit during the 15 min reaction at 37°C and pH 7. Effective α-amylase activities (SAU/s) were computed by taking the product of the salivary flow rate (mg/s) and the corresponding SAU per mg saliva.

Survey for carbohydrate consumption

Subjects were asked to fill out a questionnaire in order to estimate the amount of carbohydrate-rich food consumed at a given time period. The questionnaire resembled the Block 2005 Food Frequency Questionnaire (NutritionQuest), but included only 22 carbohydrate-rich food items (e.g. bagel, bread, crackers, pasta, potato, rice, etc.). For each food item, an approximate serving size (e.g. 1/2 bagel, 1 slice of bread, 1/2 cup cooked pasta) was also provided. The common serving sizes were adapted from web-based sources [United States Department of Health and Human Services (US Department of Health and Human Services)] as well as nutritional labels. Subjects indicated the average number of servings (e.g. 1, 2, 3) and the frequency of their consumption (i.e. everyday, 6, 5, 4, 3, 2, 1, 0 times of the food items per week). Carbohydrate consumption scores were calculated by multiplying the number of servings by frequency of consumption.

Subject screening

Once data collection was completed, the taste intensity ratings from each subject were examined. Not all subjects were able to detect the taste sensation of the given samples. Therefore, data from those subjects who rated the “other” taste of the starch samples above “barely detectable” were included in the actual data analysis ( N = 35; 12 M, 23 F). Note that 1 of the 35 subjects did not complete the carbohydrate consumption survey; the results for the carbohydrate consumption score include data from 34 subjects (11 M, 23 F).

Data analysis

Data from taste intensity ratings were log transformed prior to statistical analysis since responses on the gLMS tend to be log-normally distributed across subjects ( Green et al. 1993 , 1996 ). Repeated-measures analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) tests were performed to determine the effect of tasting time and sample preparation method. The t -test for independent samples was performed to determine if there were significant differences in intensity ratings of the stimuli between individuals with low and high effective α-amylase activity and carbohydrate consumption scores. The statistical significance criterion was set at P < 0.05. All statistical analyses were performed using Statistica 12 (StatSoft Inc., Tulsa, OK).

Part II: in vitro hydrolysis of starch

A total of 5 subjects (2 F, 3 M) between 20 and 30 years of age (mean = 25) participated in the study. These subjects were invited back to participate based on their relative α-amylase activities measured in the first experiment. Two subjects had high α-amylase activity while 3 had low activity. Inclusion/exclusion criteria and constraints for the subjects were the same as Part I. The experimental protocol was approved by the Oregon State University Institutional Review Board and complies with the Declaration of Helsinki for Medical Research. Subjects gave written informed consent and were paid to participate.

A total of 2 stimuli, prepared as aqueous solutions, were used in the experiment: 8% raw and 8% cooked corn starch (S4126, Sigma-Aldrich). Stimuli were prepared in the same manner as described in the first experiment.

All subjects came in at 9 AM and donated whole, stimulated saliva following the protocol described in Part I. To ensure that each subject’s activity was within the expected low and high range as determined in the first experiment, the saliva samples were assayed for α-amylase activity.

In vitro hydrolysis of starch

On the same day, the saliva samples and starch stimuli were separately placed in a water bath at 37°C for at least 30 min. One mL of the starch stimuli was pipetted into screw-capped glass tubes, which contain 1 mL of 8 mg/mL myo-inositol (Sigma-Aldrich) as an internal standard for HPLC analyses. Saliva, 0.75 mL, was reacted with the starch stimuli for 2, 15, and 30 s at 37°C. The volume of the saliva used was the mean amount of residual saliva found to remain in the oral cavity after swallowing (i.e. resting saliva) ( Lagerlöf and Dawes 1984 ). A constant volume of saliva was used such that differences in the amounts of hydrolysis products could be attributed to differences in α-amylase activities per mg saliva. Saliva and stimuli were incubated for the specified times prior to terminating the reaction by the addition of 5.25 mL boiling deionized water. The tubes were immediately vortexed for 2 s and immersed in a boiling water bath for an additional 15 min with occasional vortexing. Note that termination of salivary amylase reactions at the point of addition of boiling water was verified in experiments in which no increase in product concentration was observed following the addition of boiling water; in these experiments product concentration was monitored immediately following the addition of boiling water through subsequent incubation in the boiling water bath. The sample solutions were then filtered using a Whatman® 1 filter paper through a Buchner funnel and then again through a 3-kDa centrifugal filter (Amicon Ultra, EMD Millipore) for 1 h to remove biological compounds (i.e. proteins from saliva) that could interfere with the performance of the HPLC column. The resulting filtrate was diluted 2-fold with deionized water (i.e. 16-fold final dilution) prior to HPLC analysis. Control samples (i.e. 2 starch stimuli added with buffer instead of saliva) were included in all reactions and underwent the same conditions as the stimuli.

High performance liquid chromatography (HPLC)

The amounts of simple sugars (i.e., DP 1–2) and maltooligosaccharides (MOSs) DP 3–8 produced through salivary a-amylase hydrolysis were quantified using a Shimadzu Prominence UFLC HPLC system (Kyoto, Japan) and an evaporative light scattering detector (ELSD-LT II). DP 1–8 were the only saccharides that were analyzed since there are no commercially available standards for DP 9+. MOS were analyzed by injecting 20 µL of the hydrolysate-containing samples into the HPLC system where they were eluted at 0.2 mL/min at 80°C using deionized water as the mobile phase and Ag 2+ polystyrene ion-exchange guard and analytical columns (Supelcogel) as the stationary phase. The simple sugar and MOS concentrations were calculated from an internal standard curve generated from 5 levels of DP 1–8 (0.1, 0.3, 0.5, 0.7, 0.9 mg/mL) with myo-inositol (0.5 mg/mL) as the internal standard. DP 1 and 2 (i.e. glucose and maltose) were procured from Sigma-Aldrich while DP 3–8 (i.e. maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose) were procured from Carbosynth. Results are thus based on quantification of MOS corresponding to the retention times of these linear MOS standards. It should be kept in mind that salivary amylase digestion of starch is expected to yield both linear and branched maltooligosaccharides. All peaks were integrated by the LCsolution computer software (Shimadzu).

Taste perception of starch

Repeated measures ANOVA performed on the log perceived sweetness and “other” taste intensity ratings for the test stimuli showed significant effect of stimulus ( F 2, 68 = 220.6, P < 0.05; F 2, 68 = 37.8, P < 0.05, respectively) (see Figure 1 ). Tukey’s HSD test revealed that the significant effect of stimulus on sweetness ratings was derived from higher mean rating for glucose compared to raw and cooked starch samples ( Figure 1A ). Conversely, the significant effect of stimulus on “other” taste ratings resulted from lower mean ratings for glucose compared to raw and cooked starch samples ( Figure 1B ). On the other hand, no significant effect of tasting time was observed on the mean perceived sweet and “other” taste intensity ratings ( F 2, 68 = 0.06, P > 0.05; F 2, 68 = 0.06, P > 0.05, respectively).

Mean perceived sweetness and “other” taste intensities of test stimuli at 5, 15, and 35 s tasting times ( n = 35). “Other” taste was described by subjects as “cracker-”, “flour-”, “rice-”, “tapioca-”, and “starch-like”. Vertical bars represent the standard errors of the means (SEMs). Letters on the right y -axis represent the semantic labels of the gLMS: BD, barely detectable; M, moderate; W, weak.

The role of salivary α-amylase in the taste perception of starch

Similar to what was observed in our previous study ( Lapis et al. 2014 ), subjects’ salivary α-amylase activity per mg saliva (SAU/mg saliva) and salivary flow rate (mg saliva/s) showed no correlation. Hence, effective α-amylase activities (SAU/s) were calculated by multiplying α-amylase activity and salivary flow rate. The subjects were then divided into 2 groups based on effective α-amylase activity. The bottom and top 50% of the subjects were categorized as “low” and “high” groups, respectively. Results of independent t-tests showed that the “high” group had significantly higher mean effective α-amylase activity than the low groups ( t = 3.55, P < 0.05; see Table 1 ).

Mean (± SE) effective α-amylase activity (SAU/s) and carbohydrate consumption scores between groups of subjects a

a Groupings were made independently for each factor based on bottom and top 50% for low and high group, respectively. Subjects who belong to low group for effective α-amylase activity do not necessarily belong to low group for carbohydrate consumption score.

b Total n = 35; n = 17 for low group, n = 18 for high group.

c Total n = 34; n = 17 for low group, n = 17 for high group.

* P < 0.05, independent t -test.

The effect of salivary α-amylase rate of production on the taste responsiveness to starch samples was tested. Since no significant main effect of tasting time effect was observed, the taste intensity ratings were combined across the different time periods to simplify data analysis and presentation. Figure 2 shows the mean log “other” taste intensities for glucose, raw starch, and cooked starch. Results showed that the high amylase group had significantly higher “other” intensity ratings than the low group for cooked starch ( t = 3.02, P < 0.05) but not for glucose ( t = 0.48, P > 0.05) and raw starch (t = 0.61, P > 0.05).

Mean perceived “other” taste intensity of test stimuli grouped by subjects’ effective α-amylase activity (SAU/s). Letters on the top x -axis indicate subject grouping: L = low (bottom 50%, n = 17); H = high (top 50%, n = 18). Vertical bars represent the standard errors of the means (SEMs). * P < 0.05, independent t -tests between low and high groups. Letters on the right y -axis represent semantic labels of the gLMS: BD, barely detectable; W, weak; M, moderate.

The role of carbohydrate intake in the taste perception of starch

Individual carbohydrate consumption scores were calculated by multiplying the amount by the frequency of consumption of carbohydrate-rich foods listed in the survey provided to the subjects. The subjects were then divided into 2 groups based on their carbohydrate consumption scores (i.e. bottom and top 50% of the subjects corresponded to low and high groups, respectively). The subjects consisted of a total of N = 34 and thus had N = 17 each for low and high groups. Results of independent t -tests showed that the high groups had significantly higher mean carbohydrate consumption scores than the low groups ( t = 6.43, P < 0.05; Table 1 ). Note that this grouping was performed independent of the grouping for α-amylase activity. Importantly, the subjects’ carbohydrate consumption scores and their effective α-amylase activities were not significantly correlated ( r = 0.13, P = 0.44).

When the role of carbohydrate intake on “other” taste responsiveness of subjects was determined ( Figure 3 ), it was found that subjects with high carbohydrate consumption score showed significantly higher taste intensity ratings for cooked starch than those with low scores ( t = 2.71, P < 0.05), but not for glucose ( t = 1.31, P > 0.05) and raw starch ( t = 0.24, P > 0.05).

Mean perceived “other” taste intensity of test stimuli grouped by subjects’ carbohydrate consumption scores. Letters on the top x -axis indicate subject grouping: L = low (bottom 50%, n = 17); H = high (top 50%, n = 17). * P < 0.05, independent t -tests between low and high groups. Vertical bars represent the standard errors of the means (SEMs). Letters on the right y -axis represent semantic labels of the gLMS: BD, barely detectable; M, moderate; W, weak.

Effects of cooking and reaction time on in vitro hydrolysis of starch

Table 2 shows the amount of hydrolysis products (mg/mL) produced through in vitro salivary α-amylase hydrolysis of raw and cooked starch samples at 3 different reaction times. No DP 1 (i.e. glucose) was produced from both starch samples at all reaction time points similar to data found in the literature ( Whelan and Roberts 1953 ; Parrish et al. 1970 ; Robyt and French 1970 ). Repeated measures ANOVA performed on total DP 2–8 showed significant effect of cooking ( F 1, 4 = 18.0, P < 0.05) and reaction time ( F 2, 8 = 11.0, P < 0.05) but not their interaction ( F 2, 8 = 4.1, P > 0.05). Cooked starch had significantly higher amount of total DP 2–8 than raw starch at all 3 reaction times tested ( Table 2 , see total DP 2–8 across time points, P < 0.05). Results also indicated that raw and cooked starches were hydrolyzed even at 2 s as shown by the presence of DP 2–8 at this reaction time. In raw starch, amounts of total DP 2–8 showed an increasing trend with increasing reaction time, although the differences were not significant ( P > 0.05). In cooked starch, amounts of total DP 2–8 increased with increasing reaction time with those at 30 s being significantly higher than at 2 s ( P < 0.05), but not necessarily at 15 s. Digestion of the raw starch likely corresponds to saccharification of the relatively small amorphous fraction of the starch that is generated during processing. The amounts of individual saccharides produced from the hydrolysis were also examined. Results showed that there was a significant effect of cooking ( P < 0.05) on DP 5, 6, 7, and 8 but not reaction time ( P > 0.05). On the other hand, cooking had a significant effect on DP 3 and 4 only when reacted at 30 s ( P < 0.05). Meanwhile, no significant effect of cooking and reaction time was observed on DP 2 ( P > 0.05). Further, it seemed that over time, shorter chain saccharides (i.e. DP 2 and 3) were produced more than the longer chain saccharides (i.e. DP 4–8), especially in cooked starch. This is due to the fact that DP 2 and 3 are the hydrolysis end products and thus their products are built up over time, while DP 4–8 are on a steady state (i.e. they are broken down as they are utilized as substrates for α-amylase). Note that raw and cooked starch samples that were not reacted with saliva were also tested as control samples and no DP 1–8 were found (data not shown).

Mean (± SE) levels (mg/mL) of hydrolysis products produced through in vitro α-amylase hydrolysis of 8% (w/v) raw and cooked starch at 3 different reaction times

Values obtained from a total of 5 subjects; each sample was reacted with a constant volume of saliva from each subject. Sample means that have no superscripts in common within each row are significantly different from each other (Tukey’s HSD, P < 0.05). DP, degree of polymerization; ND, none detected.

* Maltooligosaccharides DP 1–8 were not detected in samples void of saliva.

** Theoretical values obtained by subtracting total DP 2–8 from 80 mg/mL, the starting concentration of the starch samples.

The effect of different levels of salivary α-amylase activity on the in vitro hydrolysis of starch

To determine the role of different levels of salivary α-amylase on the in vitro hydrolysis of starch, the subjects ( n = 5) were grouped according to their α-amylase activity. Three subjects belonged to the low group (mean ± SE α-amylase activity unit: 4.0 ± 0.4 SAU/mg saliva) while 2 belonged to high group (mean ± SE α-amylase activity unit: 24.1 ± 3.0 SAU/mg saliva). The mean total DP 2–8 (mg/mL) were then calculated and compared between subjects with low and high α-amylase activities (i.e. SAU per mg saliva) ( Figure 4 ). Results showed a trend for higher mean total DP 2–8 produced from raw starch at 2, 15, and 30 s by subjects with high α-amylase activity than with low activity, although the difference was not significant ( P > 0.05). As noted previously, the digestibility of the raw starch preparation likely reflects that this preparation has a small, but significant, amount of amorphous character. Results also showed that subjects with high α-amylase activity produced significantly higher mean total DP 2–8 from cooked starch at 15 and 30 s ( F 2, 8 = 11.0, P < 0.05) than with low activity but not at 2 s ( F 2, 8 = 11.0, P > 0.05). The mean total DP 2–8 from cooked starch from subjects with low α-amylase activity accounted for 15.5, 17.3, and 20.0% of the starting material at 2, 15, and 30 s, respectively, while those from high α-amylase accounted for 15.5, 25.8, and 31.3%, respectively.

Mean sum of DP 2–8 ± standard error generated in vitro hydrolysis. 8% (w/v) ( A ) raw and ( B ) cooked starch were incubated for 2, 15, and 30 s with saliva from subjects with low ( n = 3) or high ( n = 2) α-amylase activity. The bars represent subject grouping. Unfilled bar: L = low; filled bar: H = high. * P < 0.05, independent t -tests between low and high groups. Vertical bars represent the standard errors of the means (SEMs). Values represent the sum of DP 2–8 presented in Table 2 after dividing subjects into L and H groups based on salivary α-amylase activity unit (SAU/mg saliva). Mean ± SE α-amylase activity unit for the groups L and H were 4.0 ± 0.4 and 24.1 ± 3.0 SAU/mg, respectively.

Target substrates for taste perception of starch

The present data support the idea that starch can be tasted after it is actively mixed with saliva in the oral cavity. It is unlikely that starch itself is the stimulus for taste perception because of its bulky structure and insolubility ( Birch 1987 ; Ramirez 1991a ; Wang et al. 1998 ). During oral digestion, salivary α-amylase rapidly hydrolyzes α-1,4 glucosidic bonds in the polymeric starch chains to produce shorter chain saccharides, including maltose, linear and branched glucose oligomers, and shorter chain glucose polymers ( Robyt and French 1970 ; Jacobsen et al. 1972 ; Robyt 2008 ). These salivary α-amylase hydrolysis products appear to be critical for starch perception. (We are using the term “starch perception,” in this case, to indicate that starch is the consumed stimulus.) The regular corn starch, which was used in this study, consists of 27% amylose and 73% amylopectin. The raw starch stimulus likely has at least some amorphous character (i.e. non-crystalline regions having less molecular order); the percent amorphous character of the starch increases with cooking. The glucose units in the amylopectin molecule are mostly linked linearly (i.e. α-1,4 linkages) with branching (i.e. α-1,6 linkages) only occurring about 5% of the time (e.g. 50 branch linkages for every 1000 linkages) ( Robyt 2008 ). Also note the absence of glucose in the salivary amylase-catalyzed hydrolysis of starch, as evidenced by our in vitro digestion study (see Table 2 ). Thus, one may speculate on the target substrates that are responsible for the perception of starch.

First, it is possible that maltose contributed to the taste responsiveness to starch samples, although it is unlikely that maltose was fully responsible for the responsiveness. Current results from the in vitro digestion study showed that only 1.0–1.9 and 2.0–5.1 mg/mL of maltose were produced by α-amylase from the original concentration (80 mg/mL) of raw and cooked starches, respectively (see Table 2 ). Considering only 8% starch samples were presented to the subjects in our psychophysical study, approximately 0.1–0.2% and 0.2–0.5% of maltose might have been produced from raw and cooked starch samples, respectively. These estimated amounts are below the known detection threshold of 1.3% for maltose ( American Society for Testing and Materials 1973 ), which suggests that without accounting for potential synergistic interaction with other saccharides, maltose by itself was not the primary source of taste responsiveness to starch samples.

A second possibility is that glucose oligomers produced via salivary α-amylase hydrolysis of starch are the terminal stimuli. The presumption that humans can taste glucose oligomers is supported by our recent data showed that in the absence of olfactory and textural cues, humans could taste 75 mM glucose oligomers with average DP 7 and 14 but not glucose polymers with average DP 44 ( Lapis et al. 2016 ). Note that the definition of oligomers varies across fields of study; some consider those that contain 2–10 monomer units as oligomers ( Rocklin and Pohl 1983 ; Sclafani 1987 ), while others count monomer units up to 20 as oligomers ( Hughes and Johnson 1981 ). Unfortunately, the full hydrolysis profile of starch beyond DP 8 as well as branched oligomers was not quantified in this study because commercial HPLC standards for DP 9+ are not available for purchase.

The values of DP 9+ estimated for raw starch are calculated by difference (total starch minus DP 1–8), thus they include insoluble, unhydrolyzed starch granules along with minor amounts of the higher DP soluble hydrolysis products. It is commonly believed that only soluble stimuli cause taste receptor mediation and therefore taste responsiveness ( Birch 1987 ). Based on this premise, the DP9+ fraction is not thought likely to elicit a taste response. However, a gustatory mechanism that detects insoluble raw starch has been proposed ( Ramirez 1991a ). Findings showed rats were able to detect low concentrations of raw starch (i.e. 0.5%) suspended in water from several species of plants (i.e. corn, rice, wheat, and potato), while they ignored comparable concentrations of cellulose suspended in water ( Ramirez 1991a ). This discrimination of insoluble starch versus insoluble cellulose may, however, be the result of limited starch hydrolysis resulting from the presence of taste cell exported amylases ( Merigo et al. 2009 ); such hydrolysis would not occur for cellulose. It was found that TRPM5 knockout mice display greatly reduced starch preference, which implicates gustatory mediation of starch preference ( Sclafani et al. 2007 ). These same authors showed that gustducin KO mice have disrupted preference for Polycose (a commercial maltooligosaccharide preparation) but not starch, indicating these 2 starch-based/derived stimuli have distinct sensory signaling pathways. Furthermore evidence of this is the report that conditioned aversions of rats to starch and Polycose do not cross generalize ( Ramirez 1991b ).

The underlying sensory mechanism(s) of glucose oligomers perception has not been conclusively elucidated. Recent studies with mice have shown that some taste cells express α-glucosidases (e.g. amylase) and intestinal “brush border” disaccharide-hydrolyzing enzymes [e.g. maltase-glucoamylase (MGAM) and sucrase-isomaltase (SIS)] ( Sukumaran et al. 2016 ). A glycosidase-based mechanism, presumably dependent on hydrolysis of glucose oligomers to glucose, is supported by other data indicating selected taste cells express glucose transporters (GLUTs), sodium-glucose co-transporter 1 (SGLT1), and ATP-gated K + (K ATP ) metabolic sensors ( Yee et al. 2011 ). These findings suggest that gustatory tissues express components necessary for detection of glucose oligomers via conversion to glucose and subsequent cell uptake, analogous to nutrient sensing in the small intestine ( Zhang et al. 2015 ). Certainly, a glucose oligomer-sensing mechanism based on enzymatic hydrolysis of glucose oligomers is plausible. A second possibility to explain the taste perception of glucose oligomers is that of a novel receptor-based mechanism, as originally hypothesized to exist in rodents ( Nissenbaum and Sclafani 1987 ).

Role of other sensory cues in the detection of starch hydrolysis products

Certainly, taste is not the only sensory cue that can be used for starch detection. It can be argued that the most notable sensory property of starch is its unique texture ( Mason 2009 ), which varies greatly depending on many factors including concentration and moisture-heat treatments ( Conde-Petit 2003 ; Mason 2009 ). Starches also evoke a mild odor, potentially due to the presence of volatiles that are present in the raw materials ( Robyt 2008 ). To prevent olfactory and textural inputs into taste ratings, subjects were asked to wear nose clips and to describe texture qualities, respectively. Subjects described glucose as sweet, while they described the starch samples as “cracker-”, “flour-”, “rice-”, “tapioca”, and “starch-like” among others. These descriptors were consistent with recent data from a focus group study wherein subjects described the taste of glucose oligomers as “starchy” like a root vegetable, corn, bread, or pasta ( Lapis et al. 2016 ). There was no guarantee that every subject was able to objectively separate taste from texture, especially since the descriptors provided by subjects could also be interpreted as texture-like qualities. However, if subjects were incorporating texture with taste, it is expected that mean ratings would be much higher. In fact, the mouthfeel of the cooked starch sample is highly viscous at first (e.g. similar to a thick starch paste, thicker than pudding) then becomes thin as being tasted over time and the raw starch sample was sandy/gritty.

Effect of cooking and tasting time on the digestion and perception of starch

Results of this study showed that cooking and tasting time influenced the extent of the in vitro digestion of starch. The findings showed that cooking significantly increased the amount of hydrolysis products especially as starch is digested for a longer period of time; DP 2–8 produced from raw starch was 8.9% max compared to 24.5% max from cooked starch (computed from values in Table 2 ). In support of these findings, other studies have shown that cooking greatly increased the rate at which starch can be hydrolyzed ( Snow and O’Dea 1981 ; Slaughter et al. 2001 ; Hickman et al. 2009 ). Additionally, time course studies showed increasing trend in extent of starch hydrolysis with increasing reaction times ( Snow and O’Dea 1981 ). Previous studies showed that the thickness of starch solution decreased by 69–90% within 10 s of saliva addition and viscosity was almost non-detectable after 60 s ( Hanson et al. 2012b ). Correspondingly, time-intensity ratings of oral perception of viscosity of a starch solution showed decreasing trend over the course of 60 s ( Mandel et al. 2010 ). The rapidity with which lower DP MOSs were produced when starch was exposed to salivary amylase is noteworthy; 15.5% of cooked starch was converted to DP 2–8 within 2 s (computed from values in Table 2 ). Note that the profile and amounts of shorter chain saccharides generated by in vitro hydrolysis by expectorated saliva may differ from those generated by human oral digestion. For instance, oral digestion of starch may involve membrane-bound α-glycosidases (e.g. MGAM, SIS) ( Sukumaran et al. 2016 ), which are not present in saliva. Nevertheless, the in vitro starch hydrolysis data demonstrate the effect of cooking and reaction time on starch hydrolysis.

Results from psychophysical study showed no differences in responsiveness across sample preparation and tasting times ( Figure 1 ). This somewhat unexpected result can potentially be explained by the findings that the amounts of glucose oligomers produced by amylase hydrolysis were relatively low and further that the differences in the amounts across sample preparation and tasting times were minor. Based on our in vitro hydrolysis study, total DP 2–8 produced from 8% raw and cooked starch samples range from 0.56 to 1.96%. Such narrow range was probably not sufficient to produce a significant difference in taste responsiveness. Regardless, the concentration of starch samples used in this study was notably lower than what would be typically found in most starch-rich foods (e.g., bread, pasta, rice), which range from 13 to 87% ( Englyst et al. 1983 ). This is due to experimental constraints that at a higher concentration the starch samples were very thick and thus continuous stirring during cooking became problematic. It is likely that cooking and tasting time will have an effect on taste responsiveness at the much higher concentrations of starch that are commonly found in starch-rich foods.

The cooking parameters chosen for this work are best viewed as being an example of one possible cooking scenario. The cooked starch used in this study was held above the typically recognized gelatinization temperature range for starch in excess water (~64–75°C) ( Jane et al. 1999 ; Ratnayake and Jackson 2006 ); this treatment is consistent with an increase in granule volume and enzymatic susceptibility ( Wang and Copeland 2013 ). Following heating, starches were cooled to room temperature prior to subject testing; retrogradation is expected during this cooling phase (the term “retrogradation” herein refers to the reassociation of starch components disrupted as a result of heating; Wang et al. 2015 ). The digestibility of gelatinized starch is expected to decrease as a result of retrogradation ( Zhou and Lim 2012 ). It is important to keep in mind that starch gelatinization, pasting, and retrogradation involve complex molecular events that are dependent on many factors, including the botanical source of the starch, the processing history of the starch and the composition of the food ( Schirmer et al. 2015 ). Thus, the values obtained for the cooked starch samples in this work may not be directly/quantitatively applicable to other cooked starches.

Effect of salivary α-amylase activity on the perception and digestion of starch

The present findings demonstrated that individual differences in the salivary α-amylase activity play a significant role in the efficiency of starch hydrolysis ( Figure 4 ) and to some extent, in taste perception of starch ( Figure 2 ). Expectorated saliva with higher α-amylase activity produced significantly more hydrolysis products from cooked (but not raw) starch, than those with low activity, especially at longer tasting times ( Figure 4 ). Correspondingly, subjects with high effective amylase activity had significantly higher responsiveness to cooked but not raw starch ( Figure 3 ). Thus, individual differences in taste responsiveness to starch can be aided by salivary α-amylase activity, which dictates the final concentration of glucose oligomers. While this study focused on the effect of salivary α-amylase on taste perception of starch, it has been shown that various α-glucosidases (e.g. amylase, MGAM, SIS) are also expressed in taste cells in mice ( Sukumaran et al. 2016 ). It is yet to be seen whether similar enzymes are expressed in human taste cells and if they do, what role such enzymes play in the detection of starch hydrolysis products.

Effect of carbohydrate intake on the perception of starch

The present findings suggest that an individual’s level of dietary carbohydrate intake also influences the taste responsiveness of cooked but not raw starch. Results showed that those with high carbohydrate consumption scores had higher intensity ratings for cooked starch than those with low carbohydrate consumption scores ( Figure 3 ). The method used to collect information regarding complex carbohydrate intake was only indicative of the extent of complex carbohydrate intake per se, and does not accurately represent extent of consumption relative to other macronutrients (e.g. proteins, fats). Therefore, data could not reveal whether for example, the subjects in the high group generally eat high amounts of all kinds of foods including carbohydrates or whether they eat high amounts of carbohydrates but low amounts of other macronutrients. In the past, the differences in the level of consumption of starch-rich foods have been found to correlate with the copy number variation of salivary α-amylase gene ( AMY 1 ) ( Perry et al. 2007 ). Selected groups of people with traditionally high-starch diets (i.e. European American, Hadza, Japanese) had more AMY1 copies than those with low-starch diets (i.e. Biaka, Datog, Mbuti, Yakut) ( Perry et al. 2007 ). In the present study the subjects’ carbohydrate consumption scores and their effective α-amylase activities were not significantly correlated ( r = 0.13). The lack of correlation may reflect that all subjects consume a modern diet, especially since carbohydrate consumption data could not reveal whether the subjects truly had low or high starch diets relative to other macronutrients. That being said, it is unclear at the moment whether the sensitivity to glucose oligomers promotes the consumption of starch-rich foods or more frequent, high volume of starch-rich foods consumption increases the sensitivity to such foods. Further investigation on the relationship of factors influencing carbohydrate intake to carbohydrate taste sensitivity, including individual amylase activity, is warranted.

This work was supported by the USDA National Institute of Food And Agriculture (Formula Grant no. 2015-31100-06041).

The authors declare no competing financial interests.

The authors wish to thank Tyler Linscott for the assistance in data collection.

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School Science/Demonstrating the effects of amylases on starch

This experiment demonstrates the effects of heat and pH on the activity of enzymes.

Materials required

Saliva (contains amylases that break down starch), starch, iodine, test tubes, hot plate

Exact amounts should be determined by trial and error.

• Collect some saliva (no mucus) in a beaker. Conduct the experiment in test tubes or small beakers and do not put anything into your mouth to mix with saliva.
• Mix starch with water and boil until solution becomes clear. Cool. This step is necessary to homogenize the starch solution and increase the surface area of the starch for the enzymes to act on.
• Mix starch solution with a small drop of iodine. A positive reaction is indicated by a dark blue/black color.
• Mix starch solution with some saliva. Mix well. Keep at body temperature for several minutes (hold in hand). Amylases in the saliva break down the starch.
• Mix saliva/starch solution with a small drop of iodine. Reaction should be negative.

Here are some variations

• Alternative: Heat up saliva to denature the enzyme before mixing with starch solution. Iodine reaction should be positive for starch.
• Alternative: Mix saliva with acids or bases (e.g. lemon juice for safety) before mixing with starch. This can be used to demonstrate pH sensitivity of the amylase.
• Alternative: Mix saliva first with iodine. Then use the iodine/saliva mixture to test for starch. The iodine possibly inhibits the amylases in the saliva.
• Alternative: Conduct a Benedicts Test to test for simple sugars after starch solution has been treated with saliva. Benedict's test should be positive after addition with saliva. Test results are expected to be negative without addition of saliva or after heat denaturation or pH change of the saliva.

For a briefer demonstration of the enzymatic effect, hold a cracker in your mouth for 60 seconds. The amylases will break down the starch and the cracker will start to taste sweet due to the sugar produced.

• Book:School Science

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• CBSE Class 12
• CBSE Class 12 Biology Practical
• Study the Effect of Different Temperatures & 3 Different pH on the Activity of Salivary Amylase on Starch

Study the Effect of Different Temperatures and 3 Different pH on the Activity of Salivary Amylase on Starch

To study the effects of variation in temperature and pH levels on the activity of salivary amylase on starch.

Necessary Materials & Apparatus

• Test tubes.
• Wire gauze.
• Thermometer.
• Bunsen burner.
• Saliva solution.
• Iodine solution.
• pH tablets of 5, 6.7, 8.
• Beaker with water and a thermometer.
• 15 ml 1% starch solution + 3 ml 1% NaCl.
• 3 series of test tubes, each containing iodine solution.

Effect of Various Temperatures on the activity of salivary amylase on starch

• Divide and pour the 15 ml 1% starch solution + 3 ml 1% NaCl solution into three test tubes and name them as A, B and C.
• Pour a few ice cubes in a beaker and ensure that they stay at 5 °C.
• Transfer tube- A to the beaker with ice.
• Take two more beakers and fill them with water.
• Heat the two beakers, one up to 37 °C and the other at 50 °C.
• Ensure that the temperatures for the two beakers are constant.
• Transfer test tube B into the beaker which is set at 37 °C.
• Similarly, transfer test tube C into the beaker set at 50 °C.
• Draw 1 ml of saliva solution and add it into test tube A. Do the same for test tube B and C.
• Quickly draw a few drops using a dropper from test tube A and transfer the same to the first series of test tubes having iodine solution.
• Repeat the same: transfer a few drops from test tube B and C into the second and third series of test tubes having iodine solutions.
• Note the time as “0-minute reading” and wait 2 minutes before proceeding to the next step.
• Draw a few drops from each tube and add it to the tubes with the iodine solution. Note the change in colour.
• Repeat the experiment in intervals of 2 minutes until the colour of iodine does not change.

Effect of different pH levels on the activity of salivary amylase on starch

• Add pH tablets 5, 6.8 and 8 into test tube A, B, and C respectively.
• Now add water into a beaker and boil it by placing it on a Bunsen burner.
• Transfer all the three test tubes into boiling water.
• Use a thermometer to ensure that the temperature of this water is to be maintained at 37 °C.
• Use a dropper to transfer 1ml of saliva solution to each of the three test tubes.
• Immediately transfer a few drops from test tube A to the first series test tubes containing iodine solution.
• Repeat the same for test tube B and C, transferring the same to series 2 and 3 test tubes respectively.
• Draw a few drops from each tube and add it to the tubes with the iodine solution.
• Note the change in colour.

Observation

Effect of Various Temperatures on the activity of salivary amylase on starch: The test tube at 37 °C reaches the achromic point quickest compared to the other two. At high temperatures, the enzyme gets denatured and at low temperatures, the enzyme is deactivated. Hence, it takes more time for starch to be digested at temperatures outside 37° C.

The salivary amylase did not react in the tubes that had pH tablets of 5 and 8. It only reacted with the tube that had the pH tablet 6.8. The pH is considered acidic when it is level 5. A pH of 8 is considered to be alkaline. A pH of 6.8 s considered to be slightly acidic.

Learn more in detail about the effect of variations in Temperatures and pH on the Activity of Salivary Amylase on Starch , other related topics and experiments at BYJU’S Biology .

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Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

Observing earthworm locomotion

Practical Work for Learning

Published experiments

Investigating the effect of ph on amylase activity, class practical.

Measure the time taken for amylase to completely break down starch , by withdrawing samples at 10 second intervals and noting the time at which the solution no longer gives a blue-black colour with iodine solution (but the iodine solution remains orange). Use buffers to provide solutions at different pHs . Calculate the rate of this enzyme controlled reaction by calculating 1÷ time.

Lesson organisation

This procedure is simple enough for individuals to carry out if you have enough dimple tiles. If you choose to investigate five pHs, then groups of five students could complete the investigation by working together and pooling results.

Apparatus and Chemicals

For each group of students:.

Syringes, 5 cm 3 , 2 (1 for starch, 1 for amylase)

Iodine solution in a dropper bottle ( Note 4 )

Test tube rack

Test tube, 1 for each pH to be tested

Dimple tile or white tiles

Teat pipette

For the class – set up by technician/ teacher:

Amylase 1% (or 0.5%) ( Note 1 )

Starch 1% (or 0.5%) ( Note 2 )

Buffer solutions covering a range of pH, each with a labelled syringe/ plastic pipette ( Note 3 )

Health & Safety and Technical notes

Amylase solution and iodine solution are low hazard once made up. Wear eye protection when handling iodine solution. Hazards of buffers may vary. See CLEAPSS Recipe card or supplier’s information and see Note 3 .

Read our standard health & safety guidance

1 Amylase (See CLEAPSS Hazcard and Recipe card) The powdered enzyme is HARMFUL, but solutions less than 1% are LOW HAZARD. It is wise to test, well in advance, the activity of the stored enzyme at its usual working concentration to check that substrates are broken down at an appropriate rate. Enzymes may degrade in storage and this allows time to adjust concentrations or to obtain fresh stocks. Amylase will slowly lose activity, so it is best to make up a fresh batch for each lesson; batches may vary in activity and results collected on different days will not be comparable. The optimum temperature for your enzyme will be listed on the supplier’s label.

Using saliva: the CLEAPSS Laboratory Handbook provides guidance on precautions to take (including hygiene precautions) in order to use saliva safely as a source of amylase. This has the advantage of being cheaper, not requiring technicians to make up fresh solutions each lesson, it is directly interesting to students, and salivary amylase is reliable. It also provides an opportunity to teach good hygiene precautions – including ensuring that students use only their own saliva samples (provide small beakers to spit into); that students are responsible for rinsing their own equipment; and that all contaminated glassware is placed in a bowl or bucket of sodium chlorate(I) before technicians wash up.

2 Starch suspension – make fresh. Make a cream of 5 g soluble starch in cold water. Pour into 500 cm 3 of boiling water and stir well. Boil until you have a clear solution. Do not use modified starch.

3 Buffers: (See CLEAPSS Recipe card) If you make universal buffer it will contain sodium hydroxide at approximately 0.25 M, and should be labelled IRRITANT. Refer to other relevant Hazcards if you choose to make other buffers, or to supplier’s information if you purchase buffer solutions/ tablets. ( Note 1 )

4 Iodine solution (See CLEAPSS Hazcard and Recipe card). A 0.01 M solution is suitable for starch testing. Make this by 10-fold dilution of 0.1 M solution. Once made, the solution is a low hazard but may stain skin or clothing if spilled.

Ethical issues

There are no ethical issues associated with this procedure.

SAFETY: All solutions once made up are low hazard. Wear eye protection, as iodine may irritate eyes.

Preparation

a Check the speed of the reaction with the suggested volumes of reactants to be used – 2 cm 3 of starch: 2 cm 3 of amylase: 1 cm 3 of buffer at pH 6. Ideally the reaction should take about 60 seconds at this pH: this is the usual optimum for amylase (see note 1). If the reaction is too fast, either reduce the enzyme volume or increase the starch volume. If the reaction is too slow, increase the enzyme volume or concentration or reduce the starch volume or concentration.

Investigation

b Place single drops of iodine solution in rows on the tile.

c Label a test tube with the pH to be tested.

d Use the syringe to place 2 cm 3 of amylase into the test tube.

e Add 1 cm 3 of buffer solution to the test tube using a syringe.

f Use another syringe to add 2 cm 3 of starch to the amylase/ buffer solution, start the stop clock and leave it on throughout the test. Mix using a plastic pipette.

g After 10 seconds, use the plastic pipette to place one drop of the mixture on the first drop of iodine. The iodine solution should turn blue-black. If the iodine solution remains orange the reaction is going too fast and the starch has already been broken down. Squirt the rest of the solution in the pipette back into the test tube.

h Wait another 10 seconds. Then remove a second drop of the mixture to add to the next drop of iodine.

i Repeat step h until the iodine solution and the amylase/ buffer/ starch mixture remain orange.

j You could prepare a control drop for comparison with the test drops. What should this contain?

k Count how many iodine drops you have used, each one equalling 10 seconds of reaction time.

l Repeat the whole procedure with another of the pH buffers to be used, or pool the class results.

m Consider collecting repeat data if there is time.

n Plot a graph of time taken to break down starch against pH, or calculate the rate of reaction and plot rate against pH.

Teaching notes

This is a straightforward practical giving reliable, unambiguous results. The main errors will be in the order of mixing the enzyme/ substrate/ buffer, or a delay in sampling so that the reaction time is under-estimated or rate is over-estimated. Temperature variation affects enzyme activity, so results collected on different days are not comparable.

Health and safety checked, September 2008

http://rsc.org/Education/Teachers/Resources/cfb/enzymes.htm Royal Society of Chemistry: Chemistry for Biologists: Enzymes

A clear and thorough presentation of information about enzymes as chemical catalysts and the factors affecting their activity.

(Website accessed October 2011)