THIAMINE IN FISH AND ITS DEGRADATION DURING THERMAL PROCESSING OF SALTED-BOILED FISH Suparno ' )

The existence of different forms of thiamine in the llesh of rainbow trout (SoJnro gairdrwri) and. their losses during thermal processing have been investigated. Free thiamine, thiamine pyrophoephate, and protein-bound thiamine were found in the fish muscle, The average total thiamine content was 270 yS%. The largest proportion was found as pyrophosphate ester (80%) and the remaining smaller proportions were as freeand protein-bound forms (approximately 10% each). The thermal degradation rate of the total thiamine in fish was high, due mainly to the higher proportion of thiamine pyrophosphate which degraded more quickly than the other two forms. firere was an indication that thiamine pyrophosphate and protcin-bound thiamine were converted into free thiamine before they were finally degraded during the heating.


INTRODUCTION
Ttriamine (Vitamin 81) has been more widely studied in relation to heat treatment than many other nutrients in food.Consequently, the path- way of thiamine degradation by heat under different conditions, including the mechanism of the chemical reactions involved, has been largely known for some years (Dwivedi and Arnold,   1973).Thiamine degradation by other treatments such as gamma ray, tfV light irradiation and microwave energy have also been studied (Thomas et al.,l98l; Goldblith et al., lg68).
There are numerous factors influencing the thermal degradation of thiamine.These include temperature, time, pH, electrolyte system (buffer system), heavy metals, concentration of electro- lytes (the salt effect), non-electrolytes (such as protein and carbohydrate), forms of thiamine and their concentrations, oxidation-reduction system, and moisture content (Farrer, 1955).The effect of pH is the most important factor determining the stability of this vitamin during heating.At low pH (below 4) thiamine is very stable even at relatively high temperatures, but in slightly alkaline conditions considerable loss can occur.
Thiamine degradation in foods has received great attention for several reasons.Being the most heat unstable nutrient, its loss during heat treatment has eignificant nutritional conse- quences.On the other hand, its characteristic of susceptibility to heat has made it a good index of a given thermal treatment.Therefore the kinetic degradation data of thiamine may be used as an index of the efficacy of a thermal process (Stumbo,   1973;Mulley et al., 1975).Thiamine degradation has also an important role in the production of volatile flavour compounds.T'hese compoundc can be produced by heating, UV irradiation and Maillard-type reactions (Buttery et al.,1984; Hartman  et al., 1984; van Dort et al.,1984')In spite of a wide range of studiee on this vitamin in many foods, relatively little attention has been given to fish products.The present study is aimed at investigating all possible thiamine forms and their levels in fish flesh especially in relation to thermal processing.The information obtained is not only useful for nutritional assessment of these products, but it may also be required for designing optimum nutrient retention during thermal processing.Potential applications are in fish canning and traditional processing ofboiled fish (Suparno, lg88 The size of fish used was in the range of 300-350 g weight. Chemicals.All reagents used were of analytical reagent grade and preparation of all the solutions are described in A.O.A.C. (1980).Fungal amylase was obtained from Miles Laboratory Inc., Germany.IFR Journal Vol.II No.l, 1996   minutes.After being cooled, the pH of the solution was checked.If higher than pH 4.0, the solution was discarded.The solution was transferred into a 100 ml volumetric flask and made up to volume with distilled water.ltre solution was divided into 2 parts.One part (A) was used for total thiamine (free thiamine, thiamine.pyrophosphateand protein-bound thiamine) determination, and another part (B) was used for free plus bound thiamine determination.
Determination of Total Thiamine The digested solution (A) was supplemented with 1b% sodium acetate solution until the pH reached 4.5-5.0 and then 5 ml of 10% amylase solution was added.This solution was incubated in a water bath at 50-55"C for three hours.The time and temperature for optimum enzymic hydrolysis were further investigated.
Determination of Protein-Bound Thla- mine: 2-3 g of sample was weighed and trans- ferred to a homogenising vessel.After addition of 20 ml of 15% trichloroacetic acid (TCA) solution, the sample was filtered through an ashless and acid resistant filter paper.The precipitate was washed with two 10 ml portions of lb% TCA solution and digested with hydrochloric acid solution as described above (C) and protein-bound thiamine estimated.

RESULTS AND DISCUSSION
Deueloped Method of Analyeie (a) Sample Preparation: Fresh fish meat was chopped and macerated.The meat was used to study methods of thiamine analysis and the determination of thiamine contents in the fresh fish as the raw material for heat processing.
To study the effect of heat treatment on thiamine loss, the dressed fish were salted in2\o/o salt solution for two hours.Then the fish were wrapped in aluminium foil and heated in a laboratory steam retort.
Heating conditions were set at 121"C for heating times of 0, 10, 20, 30, 40, 50 and 60 minutes.Cooked fish meat samples were prepared by the same procedure as that for fresh meat samples.
As shown in Fig. 1, the first step in total thiamine analysis is acid digestion to liberate thiamine from the cell tissues.Purification of the digested solution is commonly employed to re- move interference by passing the solution through a column containing resin.This time consuming work can be omitted by treating the digested solution with sufficient crystalline KCI to form a saturated solution while maintaining the pH Protein-bound thiamine is commonly found in milk, yet it is rarely found in other foods (Farrer,   1955).It is easily converted into the free form during acid digestion.For this reason determina- tion of free thiamine in many foods may include protein-bound thiamine.Determination of pro- prior to acid digestion.On the other hand, in the determination of thiamine pyrophosphate, which is based on the determination of total thiamine, its accuracy depends signifrcantly on the effective- ness of the enzyme hydrolysis.The optimum for this condition should therefore be investigated.
The optimum pH for enzyme activity was initially discovered during a 3 hour incubation period at a temperature of 50oC as shown in Fig. 2. It appeared that pH 5.7 was the optimum.At higher pH values, a steady reduction of recoveries IFR Journal Vol.II No.t, t99G was observed.This may be partly due to thermal destruction at this elevated temperature, Similarly optimum temperature was found to be approximately 45"C as shown in to increase recovery by increasing the incubation time showed insignificant improvement.Much higher recoveries (between 93-95%') were obtained by enzymic hydrolysis carried out overnight at a temperature range between 30-35"C with a pH level of 4.5.Lowering temperature and pH levels for hydrolysis from the previous optimum condi- tions was intended to reduce microbial growth and high temperature effects.Both conditions of hydrolysis may be used in the determination of the thiamine by taking account the recovery figures in their calculations.

Thiarnine Contents in Fir,h
The results of the investigation show that all three forms of thiamine were present in the fish muscle of rainbow trout.as shown in Table 1.It is obvious that the majority of thiamine present in the fish muscle is in the form of thiamine pyrophosphate (approximately 80%) and at equal level of 10% each as free thiamine and protein- bound thiamine.It suggests that fish rely on thiamine as a coenzyme in their metabolism, which is similar to that commonly found in terrestrial animals (Higashi, 1961).
It is obvious that the thiamine pyrophosphate contents in fish muscle vary less than the other forms.It is suspected that the thiamine pyrophos' phate is maintained at about this level for biologi' cal activity (metabolism) of the fish.The free thiamine may be readily converted to thiamine pyrophosphate to maintain this level to the degree required for metabolism.
However, the roles of the other forms of thiamine in fish are not known.The level of thiamine content in fish depends also on the presence of thiaminase originating from the muscle tissue or from bacteria, or by other antithiamine factors.Thus storage of raw fish or the degree of freshness may influence reduction of the thiamine level in muscle tissues.It should be noted.howev- er, that some enzymes in the flesh or in bacteria may also synthesise this vitamin.
Thiamine Lp,eeee Instability of thiamine to heat treatment has been proved.The destruction of thiamine during processing salted-boiled fish is shown in Fig. 4. In this experiment, free and bound thiamines were not determined individually as they were present in much lower levels than the thiamine pyrophos- phate.The lower concentration of thiamine at the initial heating time compared to the level for fresh fish meat is due to liquor release by the salting process before heating as well as thermal effects during retort come-up heating time.Note: TPP = TT= thiamine pyrophosphate; FT = free thiamine; PBT = protein-bound thiamine; total thiamine It has been found that liquor releases contrib- uted by heavy salting for 2 hours is estimated 5.6% and thermal effect is about 11% from the dressed fish weight (Suparno, 1988).The liquor release is suspected to carry a major proportion of this soluble vitamin from fish muscle tissues at the initial heating time compared to the effect 54 of direct thermal degradation.About 33% of thiamine was lost due to salting and thermal effects in this period.The amounts of liquor released during subsequent heating periods were relatively small.The major contribution to thiamine losses during the remaining periods wae therefore mainly due to thermal degradation.
As shown in Fig. 4, thiamine pyrophosphate destruction was rapid during early heating and then slowly declined.On the other hand, the rate of degradation for total thiamine was more con' stant during the entire heating period.Interest' ingly, free thiamine and protein-bound thiamine accumulated during heating for about 30 minutes before their levels dropped again.Ttris fact sug- gests that thiamine pyrophosphate was partly converted to free thiamine during heating.Thia- mine pyrophosphate is thought to be more vulnerable to heat than free thiamine, although other findings are in disagreement on this (Farrer, 1955).t0 Hctln3 tlnc (nlnuto) Protein bound thiamine may also be converted to free thiamine before it is degraded, even though this form is considered more stable than others.Stability of protein-bound thiamine to heat treatment has also been shown in the pres- ent experiment.After heating for 50 minutes, no thiamine pyrophosphate was detected.Thus, by heating beyond 50 minutes, thiamine in the flesh exists as free and protein-bound forms.Since free thiamine is less stable than protein-bound thiamine, most probably the latter is more predomi- nant in the heated flesh.CONCLUSION (a) Fish muscle contains three forms of thia- mine, i.e. free, pyrophosphate ester and protein-bound forms.Thiamine pyrophos- phate constitutes the largest (80%) portion of thiamine present in fish muscle, and is believed to be associated with biological activity of the animal.
(b) It has been proved that the pyrophosphate ester is the most thermally vulnerable and the protein-bound is the least thermally vulnerable form of thiamine.The thiamine pyrophosphate is completely destroyed by heating the fish at 121'C for 60 minutes.During heating, the ester is partly converted into the free form before finally being degraded.
Figure 1.Determination of different forms of thiamine contents in fish.
thiamine is easily prepared by precipi- tation with trichloroacetic acid(TCA) solution Figure 2. Enzyme activity at different pH values (incubation temp.5O"C for 3 hrs).

Figure 4 .
Figure 4. Thermal degradation of different forms of thiamine in fish.

Table 1 .
Different forms of thiamine contents in Rainbow Trout muscle ergo/o\.