Journal of Dental Research

http://jdr.sagepub.com Whole Saliva, Salivary Sediment, and Supernatant as Fermenting Agents for Foods
Donald J. Beck and Basil G. Bibby J DENT RES 1961; 40; 479 DOI: 10.1177/00220345610400031401 The online version of this article can be found at: http://jdr.sagepub.com

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Whole Saliva, Salivary Sediment, and Supernatant as Fermenting Agents for Foods
DONALD J. BECK and BASIL G. BIBBY Eastman Dental Dispensary, Rochester, New York

During the course of an investigation into the fermentation of foodstuffs by saliva and the development of a new method of measuring acid production in these mixtures,1 the attention of the investigators was directed toward the relative fermentative capabilities of whole saliva, salivary sediment, and supernatant. It had previously been shown by Sreebny, Kirch, and Kesel,2 using pure sugars as substrate, that the intrinsic glycolytic activity of saliva lies in the micro-organisms, i.e., the enzymes are largely intracellular. This finding was confirmed by Hartles and Wasdell,3 again with pure sugar substrates. In addition, these latter investigators reported that, despite the fact that the supernatant from centrifuged saliva showed practically no glycolysis per se, the glycolytic activity of salivary sediment in the absence of suDernatant was reduced considerably compared with that of whole saliva. They offered no suggestion as to what the mechanism or factor in the supernatant might be, which, although having no intrinsic glycolytic powers, greatly increased glycolysis by the organisms. In view of the marked variations we noted between fermentations by whole saliva, salivary sediment, and supernatant in our early foodstuff experiments and the findings of these authors with pure sugars, it was decided to make some investigation of the problem in the present study. This investigation was designed (a) to attempt to confirm the findings of these two groups of workers, using foods (the normal bacterial substrate in the mouth) rather than pure sugars, and (b) to make some investigation of the possible nature of the glycolytic adjuvant powers of salivary supernatant. The so-called periodic neutralization technique,' using regular titrations with sodium hydroxide during the course of fermentation, was employed to measure the acid production in the various test mixtures.
EXPERIMENTAL METHODS

To compare whole saliva, salivary sediment, and supernatant, 5 per cent suspensions of chocolate cookie, salted cracker, corn flakes, chocolate ice cream, and banana were produced in a Waring Blendor. Three series of 30-ml. samples of these five food suspensions were prepared, and to each sample in the first series was added 10 ml. of whole, paraffin-stimulated, pooled saliva. For the other two series, whole saliva from the same pool was centrifuged for 30 minutes at 9000 rpm, sediment and supernatant
This investigation was supported in part by a U.S.P.H.S. research grant, D-607, from the National Institute of Dental Research, National Institutes of Health. Received for publication September 6, 1960.
479
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480 BECK AND BIBBY

Hay-June 1961 separated, and each made up to the original volume of the sample with distilled water. The second series employed 10 ml. of the suspension of salivary sediment thus formed in each sample, and the third series employed 10 ml. of the salivary supernatant. Controls consisting of distilled water plus whole saliva, distilled water plus sediment, and distilled water plus supernatant were set up. pH readings were taken for each sample, and they were then incubated at 3 70 C. for a total of 6 hours, with intermittent manual agitation. At the end of each hour of fermentation the pH was checked, and the mixtures were titrated back to the original pH with 0.1 N sodium hydroxide, using a pH meter* to check the end-point of the titration. The sum of the six hourly titrations was
J. D. Res.

taken as the total acid production in the mixtures. A 96-hour adaptation of this periodic neutralization technique with one food (salted cracker) was also employed to compare the fermentative capacities of whole saliva and sediment. Each day of the fermentation the samples were titrated at the end of the first, second, third, fourth, fifth, sixth, twelfth, and twenty-fourth hour. The fermentation mixtures consisted of 30 ml. of a 5 per cent food suspension with 5 ml. of saliva, or the sediment of 5 ml. of saliva, made up to 5 ml. with distilled water. In an attempt to explain the reduced acid production observed when foodstuffs were fermented with salivary sediment, as compared with whole saliva fermentations, two properties of whole saliva were investigated-those of buffering activity and of amylase activity. Either one or both of these properties, being removed with the supernatant, might explain the reduced acid production from sediment. A definite difference in buffering capacity between whole saliva and salivary sediment was demonstrated when 20-ml. samples of whole saliva, sediment, and supernatant were titrated, with regular 0.5-ml. increments, with 0.01 N sodium hydroxide. The pH was recorded after each increment. A similar process was carried out with another series of samples, using 0.01 N lactic acid. When the curves so obtained were plotted together, the buffering capacities of these three salivary samples could be demonstrated. In order to test the hypothesis that reduced amylase or buffering activity might reduce acid production from sediment during fermentation, an experiment to check the effect of the addition of artificial amylase and buffers to salivary sediment was run. The test foodstuff used was salted cracker, in the proportion of 30 ml. of a 5 per cent suspension to 5 ml. of saliva or other fermenting agent. To give a basis for comparison, samples consisting of food plus whole saliva and food plus sediment suspended in normal saline were used. An amylase activity which theoretically should have approximated that of whole saliva was achieved with one sample by the addition of 5 mg. of amylaset to sediment suspended in saline. A test of the combined effect of artificial amylase and buffering was made by adding 5 mg. of amylase to sediment suspended in a "synthetic saliva" basically similar to the calcifying solution of Burger, Lavine, Deane, and Sobel.4 Further tests were made in this experiment to determine whether the low acid production by sediment could be accounted for by osmotic shock to the organisms by the suspension of the sediment in distilled water (a technique which had been used in the earlier experiments) or by a physical effect of centrifugation. To study the possibility
*

Beckman Model M Glass electrode 43509. t "Taka-Diastase," Parke, Davis and Co., Detroit, Michigan.

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Vol. 40, No. 3

SALIVA AND COMPONENTS AS FERMENTING AGENTS 481

of osmotic shock to the cells, a sample composed of food plus sediment suspended in distilled water was set up, and, to test the theory that some physical effect during centrifugation might have been involved, another sample employed saliva that was reconstituted by remixing sediment and supernatant. Controls used in this experiment consisted of water plus whole saliva, water plus sediment, food plus amylase, and food plus buffer plus amylase.
RESULTS

The comparison of whole saliva, salivary sediment, and supernatant as fermenting agents is shown in Table 1 and Figure 1. The salivary fermenting agents plus water controls showed no significant change in pH. From these results it can be seen that whole saliva brought about the most rapid and complete acid production in all cases.
TABLE 1* COMPARISON OF ACID PRODUCTION FROM FIVE FOODS FERMENTED FOR 6 HOURS WITH WHOLE SALIVA, SALIVARY SEDIMENT, AND SUPERNATANT -PERIODIC NEUTRALIZATION TECHNIQUE
TITRATABLE ACIDITY (ML. 0.1 N NAOH)

Chocolate Cookie

Salted Cracker

Corn Flakes

Chocolate Ice Cream

Banana

Whole saliva . Salivary sediment... Salivary supernatant. .

10.9 2.3 2.1

19.1 3.3 3.9

15.0 0.6 1.8

15.8 5.4 3.9

15.5 0.8 2.5

* Each figure in this table represents the sum of six hourly titrations.

CHOCOLATE
COO KI E

C
A
B

SALTED CRACKER

1\

COR N FLAKES

BX

CHOCOLATE ICE CREAM

BANANA
l

HOURS mB

am l
4

~~C m
0

u123
5
10

15

20

ML. O.IN NAOH

FiG. 1.-Relative effectiveness of whole saliva (A), salivary sediment (B), and salivary supernatant (C) in fermenting five representative foods. Acid production measured by the periodic neutralization technique with hourly titrations for a total of 6 hours.

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482 BECK AND BIBBY

May-June 1961 The results achieved with salivary sediment as the fermenting agent show that, although acid production commenced simultaneously with sediment and whole saliva, considerably less acid was produced during fermentation with the former agent than with the latter. This suggests the presence of a glycolytic adjuvant in salivary supernatant. During the first 3-4 hours of the fermentation with supernatant, little or no acid was produced from any of the substrates. This is in agreement with the results of earlier workers and, presuming that the enzymes are largely intracellular, is to be expected. During this time lag, however, proliferation of the few bacteria unavoidably left in the supernatant after centrifugation was occurring. In the later stages of fermentation, this increase in bacterial population was reflected in measurable acid in the culture. Once this period was reached, acid production in supernatant was greater than that in sediment, again suggesting that some stimulatory agent was removed as part of the supernatant in the production of sediment.
J. D. Res.

TABLE 2 ACID PRODUCTION FROM FERMENTATION OF SALTED CRACKER WITH WHOLE SALIVA AND SALIVARY SEDIMENT OVER 96-HouR PERIOD-PERIODIC NEUTRALIZATION TECHNIQUE WITH EIGHT TITRATIONS EACH DAY
TITRATABLE ACIDITY (ML. 0.1 N NAOH)
Cracker+ Whole Saliva
TITRATABLE ACIDITY (ML. 0.1 N NAOH)

Cracker+ Sediment

Cracker+ Whole Saliva

Cracker+ Sediment

Day 1.......... Day 2.......... Day 3..........

25.1 19.3 18.4

15.9 13.1 11.9

Day4..........
Total acid

21.8

8.2

production..

84.6

49.1

The 96-hour adaptation of the periodic neutralization technique produced results which are summarized in Table 2. The figures in this table represent the sum of eight titrations carried out in each 24-hour period. Under these experimental conditions, using salted cracker as substrate, whole saliva is again seen to be more efficient in acid production than sediment alone. Curves demonstrating the buffering capacities of whole saliva, sediment, and supernatant are shown in Figure 2. Whole saliva and supernatant are shown to have very similar buffering capacities, but salivary sediment has little buffering capacity in either the acid or the alkaline range. Experimental results of the investigation of the various factors which were considered to play some part in the reduction of acid production by salivary sediment as compared with whole saliva are presented in Table 3 and Figure 3. The addition of amylase to sediment tended to give a slight increase in acid production over that recorded with sediment alone. The addition of buffers in the form of "synthetic saliva," as well as amylase, again gave a slight boost to acid production. However, in neither case did acid production approach that achieved with whole saliva. From the similarity of the diagrams for sediment in saline and sediment in water, we can conclude that
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Vol. 40, NO. 3

SALIVA AND COMPONENTS AS FERMENTING AGENTS 483

osmotic shock from the suspension of the organisms in water is not an important factor. The very similar diagrams for whole saliva and reconstituted saliva suggest that the process of centrifugation does not physically affect the metabolic activity of the organisms in the salivary sediment.
12I IV

10pH

-LACTIC

} ACIDI -

8r
5
4
6- 00 WHOLE SALIVA V-V SEDIMENT \

&-ASUPERNATANT

3~~~~~~~~~~~~~
10 9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 .-N NA OH-ML. LACTIC ACID-ML.

FIG. 2.-Buffering capacities of whole saliva, salivary sediment, and supernatant demonstrated by titration with NaOH and lactic acid.

FOOD + RECONSTITUTED
SALIVA

HOURS

I
0

2
5

ML.

0.1

10 N NA OH

15

FIG. 3.-Acid production from salted cracker by various salivary fermenting agents. Periodic neutralization technique, with hourly titrations for a total of 6 hours.
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484 BECK AND BIBBY

May-June 1961 Only one of the control tests (food plus amylase) showed any significant change
J. D. Res.

in pH, and this was almost certainly due to bacterial contamination early in the experiment, probably from the electrodes of the pH meter.
DISCUSSION

Our investigations into the relative fermentative capacities of whole saliva, sediment, and supernatant for foods have, on the whole, confirmed the findings reported with pure sugars. Acid production commenced simultaneously with whole saliva and, in reduced amounts, with sediment, whereas acid production with supernatant was delayed several hours. This suggests that the intrinsic glycolytic activity of saliva is located in the sediment (the micro-organisms). Support is also given in this study for
TABLE 3 ACID PRODUCTION FROM SALTED CRACKER BY VARIOUS SALIVARY FERMENTING AGENTS PERIODIC NEUTRALIZATION TECHNIQUE
TITRATABLE ACIDITY (ML. 0.1 N NAOH)

TITRATION AT

Food+

Whole Saliva Saliva

Food+ Sediment in Saline

Food+ Sediment in Saline+ TakaDiastase

Food+ Sediment in Synth. Saliva+ TakaDiastase

Food+ Sediment in

Food+
Reconstituted Saliva

Water

Hour 2 Hour 3

Hour 1

.....................

..................... .....................
..................... ..................... .....................

Hour 4 Hour 5 Hour 6

0.75 1.0 2.0 2.6 3.7 3.8

13.9

0.45
1.1 1.2

0.5 0.6 0.75

4.6

0.4

0.2 0.9

0.8 0.8

0.6
1.4

0.5

0.7

0.6
0.9 0.9

1.1 1.2 1.6

1.7 1.6
6.9

1.3

0.7 1.2 2.0 3.4 3.6 3.9

14.8

Total acid production...

5.4

4.9

the concept of Hartles and Wasdell3 that salivary supernatant contains an adjuvant, in the absence of which the metabolic processes are greatly retarded. These workers5 also found, as we did, that the reconstitution of saliva after centrifugation restored its activity. Our efforts to identify the adjuvant in supernatant are inconclusive. We must conclude either that amylase and buffering are not the sole factors involved or that our artificial additions did not adequately imitate these factors as they occur in whole saliva. This experiment may have been too limited, testing as it did just one amylase sample and one buffering system. It might be rewarding if attempts were made to find artificial supplements to sediment that were more representative of the naturally occurring systems in whole saliva by testing the effect of different amylase samples in different concentrations and the effect of different buffering systems.
SUMMARY

The relative fermentative capabilities of whole saliva, salivary sediment, and salivary supernatant upon foods were examined, using the periodic neutralization technique. In no case did sediment or supernatant produce as much acid as did whole
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SALIVA AND COMPONENTS AS FERMENTING AGENTS 485

saliva. The intrinsic glycolytic activity of saliva was shown to rest in the sediment, but the supernatant appeared to contain some adjuvant which increased the glycolytic potential of the organisms. In an effort to identify this adjuvant, artificial amylase and buffer systems were added to salivary sediment. While boosting acid production, these artificial additions were not so effective as natural supernatant in restoring sediment to the full glycolytic activity of whole saliva.
REFERENCES

1. BECK, DONALD J., and BIBBY, BASIL G. Acid Production during the Fermentation of Starches by Saliva, J. D. Res., in press. 2. SREEBNY, LEO M., KIRCH, E. R., and KESEL, R. G. The Location of the Glycolytic Enzymes in Saliva, J. D. Res., 29:506, 1950. 3. HARTLES, R. L., and WASDELL, MARIE R. The Metabolism of the Oral Flora. 4. The Invertase

Activity of Mixed Human Saliva, Brit. D. J., 97:231, 1954.

4. BURBER, M., LAVINE, L. S., DEANE, B. C., and SOBEL, A. E. Calcification. XIX. Calcification of

Transplanted Rachitic Bone, Proc. Soc. Exptl. Biol. Med., 96:147, 1957.
5. HARTLES, R. L., and WASDELL, MARIE R. A Comparison of the Metabolic Activity of Salivary Sediment with That of Whole Saliva, J. D. Res., 34:784, 1955 (Abstr.).

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